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Table of Contents
Genomic DNA Purification 2
Overview
Wizard
SV technology
introduces membrane-based purification for manual or
automated procedures, and the Wizard
Genomic DNA
Purification Kit provides a versatile solution-based
system for the manual isolation of gDNA from all the
above materials and from bacterial and yeast cells. This
gentle, solution-based method produces DNA suitable for
amplification, Southern blotting and genomic cloning.
Look for these symbols to find the right system
for your application.
Blood
Cultured Cells
Animal Tissue
Plant Tissue
Bacteria
Yeast
0.050.2ml 0.210ml
Manual
MagneSil ONE,
Fixed Yield Blood
Genomic System
3
9
8
1
M
A
0
2
_
3
A
Automated
Wizard
Genomic
DNA Purification Kit
MagneSil Blood
Genomic, Max
Yield System
Blood
Please Note:
In the flowcharts that follow, DNA purification
systems are listed by application (blood, cultured
cells, tissue, etc.) based on the protocols given in
the Technical Manuals provided with each system.
Applications for each system with other gDNA
sources may exist (e.g., protocols for DNA
purification from blood using the Wizard
SV
Genomic DNA Purification System). Please contact
Promega Technical Services if you have questions.
techserv@promega.com
B
u
f
f
y
c
o
a
t
o
r
w
h
it
e
b
lo
o
d
c
e
lls
?
U
s
e
c
u
lt
u
r
e
d
c
e
lls
p
r
o
t
o
c
o
ls
.
S
olution-based, totally
scalable system
. U
ses
centrifugation. S
pecific
protocols provided for
30
0
l, 3m
l and 10
m
l of
fresh w
hole blood.
Requires automation.
Magnetic-based system.
Handles 200l of blood.
Maximizes yield
from sample.
R
equires autom
ation. M
agnetic
-
based system
. D
esigned to capture ~
1g
gD
N
A
from
5
0
60
l of blood.
E
lim
inates need to quantitate after purification.
www.promega.com techserv@promega.com
3
Genomic DNA Purification
Manual
Wizard
SV 96
Genomic DNA
Purification System
3
9
8
2
M
A
0
2
_
3
A
Automated
Wizard
Genomic
DNA Purification Kit
Wizard
SV
Genomic DNA
Purification System
3
9
8
5
M
A
0
2
_
3
A
Manual
Wizard
Genomic
DNA Purification Kit
Manual
You supply
lyticase and
50mM EDTA
You supply
lysozyme and/or
lysostaphin
Gram-
negative
Gram-
positive
Cultured
Cells
Yeast Bacteria
S
o
l
u
t
i
o
n
-
b
a
s
e
d
,
t
o
t
a
l
l
y
s
c
a
la
b
le
s
y
s
t
e
m
.
U
s
e
s
c
e
n
t
r
if
u
g
a
t
i
o
n
.
P
r
o
d
u
c
e
s
h
i
g
h
m
o
le
c
u
la
r
w
e
i
g
h
t
(
>
5
0
k
b
)
g
D
N
A
s
u
i
t
a
b
le
f
o
r
a
n
y
a
p
p
l
i
c
a
t
i
o
n
,
i
n
c
l
u
d
i
n
g
P
C
R
a
n
d
S
o
u
t
h
e
r
n
b
lo
t
t
i
n
g
.
U
se
w
ith
a
m
ic
roc
e
ntrifug
e
or vac
uum
m
anifold
and
vac
uum
ad
apte
rs. G
e
t P
C
R
-
re
ad
y
g
D
N
A
in 2
0
m
inute
s
afte
r lysis. C
an proc
e
ss up
to 5
x 10
6
c
e
lls/pre
p.
Perform 96 gDNA preps
at once. Requires a vacuum
manifold. Can be used on
benchtop or automated.
Comprised of SV
membranes arrayed in a
96-well format. Can
handle up to 5 x 10
6
cells/well.
S
olution-
base
d
,
totally
sc
alable
sy
ste
m
w
ith
protoc
ols
for
bac
te
ria
and
y
e
ast.
U
se
s
c
e
nt
rifug
ation.
P
rod
uc
e
s
h
ig
h
m
ole
c
ular
w
e
ig
h
t
g
D
N
A
suitable
for
any
applic
ation,
inc
lud
ing
P
C
R
and
S
outh
e
rn
blotting
.
Promega DNA Analysis Notebook
4
Genomic DNA Purification
Genome Size Molecules/g
Human 2.9Gb 3.13 10
5
Mouse 2.7Gb 3.35 10
5
Rat 2.8Gb 3.26 10
5
Arabidopsis 3.0Gb 3.04 10
5
Tobacco 4.4Gb 2.07 10
5
Corn 2.5Gb 3.65 10
5
Wheat 16.0Gb 5.70 10
4
Yeast (S. cerevisiae) 13.5Mb 6.75 10
7
Bacteria (E. coli ) 4.7Mb 1.94 10
8
Gb = Gigabases (1 10
9
). Mb = Megabases (1 10
6
).
Manual
Wizard
Magnetic
96 DNA
Plant System
3
9
8
4
M
A
0
2
_
3
A
Automated
Wizard
Genomic
DNA Purification Kit
Requires a 96-well
grinding apparatus
Requires liquid
nitrogen, mortar & pestle
Plant
Tissue
S
olution-
base
d
, totally
sc
alable
syste
m
for plant
tissue. U
se
s
c
e
ntrifug
ation. P
rod
uc
e
s
h
ig
h
m
ole
c
ular w
e
ig
h
t
(>
5
0
k
b) g
D
N
A
suitable
for any applic
ation,
inc
lud
ing
P
C
R
and
S
outh
e
rn blotting
.
M
agnetic-based system
that can be used
in m
anual or autom
ated form
at. Purifies
gD
N
A
from
seeds or tissues. Processes
8
m
m
leaf punches, 1
5
seeds. R
equires a
M
agnaB
ot
9
6 M
agnetic S
eparation D
evice (purchase separately).
Dont see your DNA sample
type in these flowcharts?
Contact Promega Technical
Services for advice at:
techserv@promega.com
www.promega.com techserv@promega.com
5
Genomic DNA Purification
Manual
Wizard
SV 96
Genomic DNA
Purification System
3
9
8
3
M
A
0
2
_
3
A
Automated
Wizard
Genomic
DNA Purification Kit
Wizard
SV
Genomic DNA
Purification System
Overnight
proteinase K
digestion
(you supply the enzyme)
Overnight
proteinase K
digestion
(you supply the enzyme)
Animal
Tissue
P
e
rf
o
r
m
s
9
6
g
D
N
A
pr
e
p
s
a
t
o
n
c
e
.
U
se
s
a
v
a
c
uum
m
a
n
if
old
.
C
a
n
b
e
use
d
o
n
t
h
e
b
e
n
c
h
top
o
r
a
uto
m
a
te
d
.
C
o
m
pr
ise
d
o
f
S
V
m
e
m
b
r
a
n
e
s
a
r
r
a
y
e
d
in
a
9
6
-
w
e
ll
f
o
r
m
a
t.
P
r
o
c
e
sse
s
up
to
2
0
m
g t
issue
/
w
e
ll.
Use with a
microcentrifuge or vacuum
manifold with vacuum
adapters. Get PCR-ready
gDNA in 20 minutes
after lysis. Can process up
to 20mg tissue/prep.
S
o
lu
t
io
n
-
b
a
s
e
d
,
t
o
t
a
lly
s
c
a
la
b
le
s
y
s
t
e
m
.
U
s
e
s
c
e
n
t
r
if
u
g
a
t
io
n
.
P
r
o
d
u
c
e
s
h
ig
h
m
o
le
c
u
la
r
w
e
ig
h
t
(
>
5
0
k
b
)
g
D
N
A
s
u
it
a
b
le
f
o
r
a
n
y
a
p
p
lic
a
t
io
n
,
in
c
lu
d
in
g
P
C
R
a
n
d
S
o
u
t
h
e
r
n
b
lo
t
t
in
g
.
Promega DNA Analysis Notebook
6
Genomic DNA Purification
Wizard
Genomic DNA
Purification Kit
Need a versatile genomic DNA purification system?
Get it all with the Wizard
SV and SV 96
Genomic DNA Purification
Systems
The Wizard
FX or Biomek
SV Lysis Buffer.
Centrifuge.
Bind DNA.
Wash, removing
solution by
centrifugation
or vacuum.
Transfer spin
column to a 1.5ml
microcentrifuge
tube (not provided).
Centrifuge.
Mouse tail clipping
or tissue sample
Proteinase K
(Cat.# V3021)*
digestion in
Digestion Solution.
Genomic DNA
Purification from
Mouse Tail Clipping
or Tissue Sample
Genomic DNA
Purification from
Tissue Culture Cells
Wash tissue
culture cells
with 1X PBS.
Incubate at 55C
overnight
(1618 hours).
Add Wizard
SV Lysis Buffer.
Transfer lystate to minicolumn.
Elute genomic DNA.
Vacuum Adapter
(Cat.# A1331)*
Vac-Man
Laboratory
Vacuum Manifold
(Cat.# A7231)*
Wizard
SV 96
Genomic DNA Purification System. PCR products were amplified from 1l of purified
sample from each well for mouse IL-1 (1.2kb). No product is expected from wells
containing water. For further details, see Grunst, T. and Worzella, T. (2002) Introducing the
Wizard
g
)
Method of Purification
SV 96 SV Vacuum SV Spin
M C +C C4 C5 C6 C7 C8 D4 D5 D6 D7 D8 M E4 E5 E6 E7 E8
3
7
1
2
T
A
0
4
_
2
A
Mouse Tail Clippings
Water Only
1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
D
E
F
G
H
Wizard
SV 96 Genomic DNA
Purification System
Cat.#: A2370 (1 96 preps)
A2371 (4 96 preps)
Protocol:
www.promega.com/tbs/tb303/tb303.html
The Wizard
FX, Biomek
2000, or MultiPROBE
II HT/EX.
For more information visit:
www.promega.com/automethods/
3
4
4
6
C
A
0
6
_
1
A
W
ork
w
ith the term
inal 2cm
of
m
ouse tails. A
ny higher up on the tail
and you'll get m
ore connective tissue,
cartilage and bone than nucleated cells.
T
his m
aterial not only clogs colum
ns,
but few
er cells m
eans less gD
N
A
.
R
o
b
o
t
t
u
r
n
s
i
n
t
o
a
g
e
n
o
m
i
c
D
N
A
m
a
c
h
i
n
e
!
www.promega.com techserv@promega.com
9
Genomic DNA Purification
MagneSil Blood Genomic,
Max Yield & MagneSil ONE,
Fixed Yield Blood Genomic
Promega has developed two new genomic DNA
isolation systems to streamline your blood-to-analysis
pathwayMagneSil Blood Genomic, Max Yield
(a)
and
MagneSil ONE, Fixed Yield Blood Genomic
(a)
. Both
systems are designed for automated gDNA purification
on liquid-handling workstations such as the Biomek
FX.
The Max Yield System purifies 46g of gDNA from
200l of whole blood. The MagneSil ONE System
purifies 1g gDNA (50%) from 5060l whole blood.
Both systems produce gDNA that is ready for use in
PCR or multiplex amplification reactions.
MagneSil ONE, Fixed Yield Blood Genomic System
Cat.#: MD1370 (1 96 preps)
Protocol:
www.promega.com/tbs/tb313/tb313.html
For more information on automated methods visit:
www.promega.com/automethods/
MagneSil Blood Genomic, Max Yield System
Cat.#: MD1360 (1 96 preps)
Protocol:
www.promega.com/tbs/tb312/tb312.html
For more information on automated methods visit:
www.promega.com/automethods/
Purify up to 36g
gDNA from 200l
whole blood.
DNA Yields (ng) From 60l Whole Blood Samples Using
the MagneSil ONE, Fixed Yield Blood Genomic System.
Donor 1 Donor 2 Donor 3 Donor 4 Donor 5
Sample 1 1078 1078 1231 1451 1025
Sample 2 990 1092 1256 1078 998
Sample 3 970 1047 1315 990 994
Sample 4 1209 1294 1047 970 967
Sample 5 1105 1063 1388 1209 843
Sample 6 839 843 1296 1105 797
Mean 1032 1070 1256 1134 937
SD 128 143 116 178 94
4
0
3
1
C
A
0
3
_
3
A
Analysis of DNA isolated using the MagneSil Blood Genomic, Max Yield System. DNA isolated from whole blood using the MagneSil Blood Genomic, Max Yield System was
used with the PowerPlex
16 System (Cat.# DC6531), a multiplex STR amplification system for use in DNA typing. Results show successful coamplification and 3-color detection of the 16 loci
(15 STR loci plus amelogenin) in the PowerPlex
16 System
(b,c,d)
. Amplification products were separated on an ABI PRISM
Software.
Purify ~1g gDNA
from 5060l
whole blood.
F
ixe
d
Y
ie
ld
m
e
an
s
n
o
m
ore
quan
titation
or
n
or
m
aliz
ation
!
O
ptim
iz
e
t
h
e
v
olum
e
of
e
lute
d
D
N
A
y
ou
n
e
e
d on
c
e
use
t
h
at
sam
e
am
oun
t
e
ac
h
tim
e
.
Wizard
Magnetic 96 DNA
Plant System
The Wizard
2000,
Biomek
Genomic DNA Purification Kit 100 isolations (300l blood per isolation) A1120
500 isolations (300l blood per isolation) A1125
100 isolations (10ml blood per isolation) A1620
For Laboratory Use. Cat.# A1120 will give ~40 animal tissue preps, ~80 mouse tail preps, ~80 plant tissue preps, and ~80 cultured cell preps. Please see the Wizard
SV Genomic DNA Purification System 50 preps (20mg tissue per prep) A2360
250 preps (20mg tissue per prep) A2361
Vac-Man
SV Genomic DNA Purification System can be used in spin or vacuum format. The Vac-Man
SV 96 Genomic DNA Purification System 1 96 preps (20mg tissue per prep) A2370
4 96 preps (20mg tissue per prep) A2371
Vac-Man
96 Magnetic Separation
Device and the MagnaBot
Spacer, 1/8 inch, are also required for use with these systems.
Product Size Cat.#
Wizard
Vent
Deep
Characteristic AmpliTaq
Platinum
Pfu
>95% >95%
Resulting DNA ends 3 A 3 A 3 A 3 A Blunt Blunt Blunt
53 exonuclease activity Yes Yes Yes Yes No No No
35 exonuclease activity No No No No Yes Yes Yes
Want to explore the enzymology of Thermostable
DNA Polymerases more thoroughly?
Go to our online Polymerase Guide at:
www.promega.com/guides/
}
U
sing
a
P
roofre
ad
ing
Polym
e
rase
?
P
roofre
ad
ing
polym
e
rase
s
w
ork
a
little
slow
e
r
th
an
non-
proofre
ad
ing
polym
e
rase
s.
B
e
sure
to
inc
re
ase
th
e
e
xte
nsion
tim
e
to
at
le
ast
2
m
inute
s
pe
r
k
ilobase
.
A
lso,
you
m
ay
ne
e
d
2
-
3
m
ore
c
yc
le
s.
Optimization of PCR
Magnesium Concentration
Magnesium concentration is an important factor to
optimize when performing PCR. The optimal Mg
2+
concentration varies depending on the primers,
template, DNA polymerase, dNTP concentration and
other factors. Taq DNA polymerase is the most common
polymerase used for PCR. Taq has an optimal Mg
2+
range of 14mM MgCl
2
. Other polymerases may have
different optimal ranges. For example, Tth DNA
polymerase has a narrower optimal range (1.52.5mM
MgCl
2
), Tli DNA polymerase displays optimal activity at
26mM MgCl
2
, and Pfu DNA polymerase has an optimal
range of 26mM MgSO
4
. Tfl DNA polymerase has an
optimal range of 14mM Mg
2+
but performs better with
MgSO
4
than with MgCl
2
.
When using a pair of PCR primers for the first time, it is
advisable to perform a magnesium titration in 0.5 or
1.0mM increments to determine the optimal Mg
2+
concentration. Some primers will amplify equally well at
a number of Mg
2+
concentrations, while others may
have very specific Mg
2+
concentration requirements.
With too little Mg
2+
, the polymerase will have poor
activity. With too much Mg
2+
, nonspecific amplification
can become a problem. Nonspecific PCR products can
appear as a smear on a gel or as distinct bands of
inappropriate size. Too much Mg
2+
can also reduce the
fidelity of the DNA polymerase and lead to a higher
error rate.
Taq DNA polymerase is commonly supplied with buffers
containing a fixed concentration of Mg
2+
(giving a final
concentration of 1.5mM in the final reaction). Most Taq
DNA polymerase amplifications work well at this Mg
2+
concentration, and the reaction can still be optimized by
adding more Mg
2+
. Pfu DNA polymerase does not have
as great a dependence upon Mg
2+
and is most often
supplied with a buffer containing a final concentration of
2mM Mg
2+
. However, this does not mean that
optimization is unnecessary, and the final concentration
of Mg
2+
can be adjusted up to 6mM as needed.
Primer Design
PCR primers generally range from 1530 bases long
and are designed to flank the DNA region of interest.
Primers should have 4060% GC content, and care
should be taken to avoid sequences that might produce
intermolecular or intramolecular secondary structure. To
avoid the production of primer-dimers, the 3-ends of
the primers should not be complementary. Primer-
dimers unnecessarily sequester primers away from the
reaction and result in an unwanted polymerase reaction
that competes with the desired PCR product. Avoid
three G or C nucleotides in a row near the 3-end of the
primer, as this may result in nonspecific primer
annealing, increasing the synthesis of undesirable
products. Ideally, both primers should have nearly
identical melting temperatures (T
m
), allowing both
primers to anneal roughly at the same temperature. The
annealing temperature of the reaction is dependent upon
the primer with the lowest melting temperature. For
assistance with calculating the T
m
of any primer, a T
m
calculator is provided on the Promega web site at:
www.promega.com/biomath
Promega DNA Analysis Notebook
14
Amplifying DNA
Vortex all MgCl
2
-containing solutions
thoroughly prior to use!
MgCl
2
solutions can form a
concentration gradient upon thawing.
Failure to vortex is a common source
of PCR failure!
N
e
e
d
to
c
a
lc
ula
te
y
o
u
r
T
m
?
G
o
to
P
rom
e
g
a's
B
ioM
at
h
pag
e
at
:
w
w
w
.prom
e
g
a.c
om
/biom
at
h
T
h
e
p
r
o
g
r
a
m
r
e
t
u
r
n
s
r
e
s
ult
s
f
r
o
m
t
h
r
e
e
d
if
f
e
r
e
n
t
p
u
b
lis
h
e
d
m
e
t
h
o
d
s
f
o
r
T
m
c
a
lc
ula
t
io
n
.
Y
o
u
c
a
n
a
ls
o
se
le
c
t
P
r
o
m
e
g
a
p
r
im
e
r
s
t
o
e
xa
m
in
e
t
h
e
ir
T
m
a
n
d
d
e
t
e
r
m
in
e
t
h
e
T
m
o
f
y
o
u
r
o
w
n
p
r
im
e
r
s
in
d
if
f
e
r
e
n
t
r
e
a
c
t
io
n
b
uf
f
e
r
s
.
www.promega.com techserv@promega.com
15
Amplifying DNA
Optimization of PCR (continued)
Annealing Temperature
Annealing temperature is another factor that may need
to be optimized in PCR. The melting temperature (T
m
) of
the PCR primers should be in the range 4265C,
unless the primers fall into a special class, such as
degenerate primers, which have lower T
m
. Typically the
optimal annealing temperature is 5C of the primer
with the lowest T
m
. Ideally the T
m
of both primers will be
similar so that the optimal annealing temperatures are
close. If the melting temperatures are more than a few
degrees apart, one primer may need to be redesigned so
that the T
m
is closer to that of the other primer. A good
starting point is to set the annealing temperature equal
to the T
m
of the primers. If nonspecific amplification
occurs, this is a good indication that the annealing
temperature needs to be raised a few degrees. If the
PCR reaction yields no product, this may indicate that
the annealing temperature is too high and should be
reduced by several degrees.
Template Quantity
The amount of template required for successful
amplification is dependent upon the complexity of the
DNA sample. For example, in a 4kb plasmid containing a
1kb insert, 25% of the input DNA is the target of
interest. Conversely, a 1kb gene in the human genome
(3.3 10
9
bp) represents approximately 0.00003% of
the input DNA. Approximately 1,000,000-fold more
human genomic DNA is required to maintain the same
number of target copies per reaction. Two common
mistakes are the use of too much plasmid DNA or too
little genomic DNA. If possible, start with up to 10
4
copies of the target sequence to obtain a signal in
2530 cycles, but do not exceed 10ng/l (i.e.,
500ng/50l reaction).
Template Quality
The purity and integrity of the DNA template can also be
critical. Obviously there are numerous inhibitors that can
interfere with amplification. These may be copurified
from the original source of the nucleic acid (e.g., the
tissue from which the DNA was isolated). Contaminants
can also be introduced during the purification process.
Examples of common contaminants that can inhibit PCR
are phenol, ethanol, as little as 0.01% SDS or other
detergents, heparin and salts. These contaminants can
usually be removed by a simple phenol:chloroform
extraction followed by ethanol precipitation, or by use of
a PCR clean-up system (see Chapter 3). Some sample
types, such as blood, soil, fungus, plants with high
phenolic content, and fecal samples, are problematic
because they contain strong PCR inhibitors that can be
copurified with the DNA. An easy way to identify
inhibitors in your template nucleic acid is to add an
aliquot of template to the positive control reaction. If
this spiked control reaction fails, the template needs to
be further purified before amplification.
Test template quality by adding a control template that
you know amplifies easily and reliably. Combine your
problematic template with 1001,000 copies of the
control template, and amplify the control template.
Perform the same amplification with the control
template alone. If amplification of the control template
fails only when the problematic template is present,
inhibitors in the problematic template may be to blame.
R
e
ac
tion
anne
aling
te
m
pe
rature
sh
ould
be
5
C
of
th
e
T
m
of
th
e
P
C
R
prim
e
r
th
at
h
as
th
e
low
e
st
T
m
.
H
ow
M
any M
ole
c
ule
s in Your
D
N
A
T
e
m
plate
?
1g
of 1k
b d
sD
N
A
=
9
.12
x 10
11
m
ole
c
ule
s.
1g
of pG
E
M
V
e
c
tor D
N
A
=
2
.8
5
x 10
11
m
ole
c
ule
s.
1g
of lam
bd
a D
N
A
=
1.9
x 10
10
m
ole
c
ule
s.
1g
of E
. c
oli g
e
nom
ic
D
N
A
=
2
x 10
8
m
ole
c
ule
s.
1g
of h
um
an g
e
nom
ic
D
N
A
=
3
.0
4
x 10
5
m
ole
c
ule
s.
Promega DNA Analysis Notebook
16
Amplifying DNA
Optimization of PCR (continued)
PCR Enhancers
In some cases it may be helpful to add certain enhancing
agents to a PCR, despite all other attempts to optimize
conditions. Two good examples are the amplification of
GC-rich templates and amplification of templates that
form strong secondary structures, which can cause DNA
polymerases to stall. GC-rich templates can be
problematic due to inefficient separation of the two DNA
strands or because of the tendency of GC-rich primers to
form intermolecular and intramolecular secondary
structures that compete with template annealing. There
are many PCR-enhancing agents that act through a
number of different mechanisms. PCR-enhancing
reagents will not work with all reactions; the beneficial
effects are often template- and primer-specific.
Betaine, DMSO and formamide can be helpful when
amplifying GC-rich templates. Betaine reduces the
amount of energy required to separate the strands of a
GC-rich DNA template (1). Dimethylsulfoxide (DMSO)
and formamide are thought to aid in the amplification of
GC-rich templates in a similar manner by interfering
with the formation of hydrogen bonds between the two
strands of DNA (2). Some reactions that amplify poorly
in the absence of enhancers will give a strong PCR
product when betaine (1M), DMSO (110%), or
formamide (15%) are added to the reaction. DMSO
concentrations greater than 10%, and formamide
concentrations greater than 5% will cause inhibition of
Taq DNA polymerase and, presumably, other DNA
polymerases as well. (3).
In some cases, general stabilizing agents such as BSA
(0.1mg/ml), gelatin (0.11.0%), and nonionic detergents
(00.5%) can overcome failure to amplify a region of
DNA. These additives can increase the stability of the
DNA polymerase and may also coat the sides of the PCR
tubes so that reagents are not lost through adsorption
to the tube walls. BSA has also been shown to
overcome the inhibitory effects of melanin on RT-PCR
(4). Nonionic detergents, such as Tween
-20, NP-40,
and Triton
-T & pGEM
-T Easy Systems
(h,i)
A1360, A1380, A3600, A3610 Yes Yes
pTARGET Mammalian Expression Vector System
(i,j)
A1410 Yes Yes
PCR Clean-Up
Wizard
X-100 at
the 1X concentration. Taq DNA Polymerase in Storage
Buffer B does not have this requirement and can be
used either with the supplied Promega Reaction Buffer
or with other Taq DNA polymerase reaction buffers.
PCR Core System I
Cat.#: M7660 (200 50l reactions; 1.25u Taq DNA
Polymerase/reaction)
Comes with 250u Taq DNA Polymerase in Storage
Buffer B, Taq DNA Polymerase 10X Reaction Buffer
without MgCl
2
, Taq DNA Polymerase 10X Reaction
Buffer with MgCl
2
(1.5mM at 1X), 25mM MgCl
2
, PCR
Nucleotide Mix.
PCR Core System II
Cat.#: M7665 (200 reactions; 1.25u Taq
Polymerase/50l reaction)
Same components as M7660 plus Positive Control
Plasmid DNA template and Upstream and Downstream
Control Primers.
Protocol:
www.promega.com/tbs/tb254/tb254.html
Taq DNA Polymerase in Storage Buffer A
Cat.#: M1861 (100u; 80 reactions)
M1865 (500u; 400 reactions)
M1868 (2,500u; 2,000 reactions)
Supplied with Taq DNA Polymerase 10X Reaction
Buffer, 25mM MgCl
2
. One reaction uses 1.25u of
enzyme.
Taq DNA Polymerase in Storage Buffer B
Cat.#: M1661 (100u; 80 reactions)
M1665 (500u; 400 reactions)
M1668 (2,500u; 2,000 reactions)
Supplied with Taq DNA Polymerase 10X Reaction
Buffer, 25mM MgCl
2
. One reaction uses 1.25u of
enzyme.
Citations for use of Taq DNA Polymerase online at:
www.promega.com/citations/
3
9
8
7
M
A
0
2
_
3
A
Primers
Template
All components
supplied in
PCR Core Systems
Taq DNA
Polymerase
dNTPs
10X Thermophilic
Reaction Buffer
MgCl
2
T
h
e
P
C
R
C
o
r
e
S
y
s
t
e
m
s
a
r
e
g
r
e
a
t
f
o
r
r
e
s
e
a
r
c
h
e
r
s
ju
s
t le
a
r
n
in
g
P
C
R
See p. 12 for
typical reaction
set-up.
S
e
e
p
.
2
3
f
o
r
d
N
T
P
s
.
www.promega.com techserv@promega.com
21
Amplifying DNA
Proofreading Polymerases
Incorporation fidelity can be an important consideration
for cloning projects. Thermostable enzymes with a
35 exonuclease activity, commonly known as
proofreading activity, offer the highest fidelity in
amplification reactions. Proofreading enzymes like Pfu
and Tli DNA Polymerase
(e)
offer three- to sixfold higher
fidelity than standard Taq DNA Polymerase. In general,
proofreading enzymes extend primers a little slower
than Taq DNA Polymerase and thus typically require
longer extension times and a few more cycles. Assume
2 minutes per kilobase of amplimer, and add 23 cycles
to your reaction when using a proofreading enzyme.
With Pfu DNA Polymerase
(e)
, it is important to use the
Reaction Buffer supplied with the enzyme for maximum
fidelity. Pfu Reaction Buffer is formulated to give
maximum fidelity, not maximum yield. After all, you use
Pfu for fidelity not yield. If you need greater yield, use
more template DNA.
Reference
1. Cline, J., Braman, J.C. and Hogrefe, H.H. (1996) PCR fidelity of Pfu DNA
polymerase and other thermostable DNA polymerases. Nucl. Acids Res. 24,
354651.
Comparison of sources of native Pfu DNA Polymerase. A 1.2kb fragment of human
1-antitrypsin gene was amplified using Pfu DNA Polymerase from Promega (Panel A) and
from another supplier (Panel B). The target was amplified from decreasing amounts of Human
Genomic DNA (Cat.# G3041) as indicated. Lane M, 100bp DNA Ladder (Cat.# G2101).
Accuracy of thermostable polymerases. The accuracy of Pfu DNA Polymerase has
been reported by Cline et al. as 7.7 10
5
. Using the PCR-based forward mutation assay,
they reported the accuracy of Pfu as approximately twofold higher than Vent
(Tli DNA
Polymerase) and approximately sixfold higher than Taq DNA Polymerase (1).
Pfu DNA Polymerase*
Cat.#: M7741 (100u; 80 reactions)
M7745 (500u; 400 reactions)
Each provided with enzyme (5u/l), Pfu DNA
Polymerase 10X Reaction Buffer (2mM MgSO
4
@ 1X)
sufficient to give the indicated number of 50l
reactions using 1.25u of enzyme.
Protocol:
www.promega.com/tbs/9pim774/9pim774.html
Citations for use of Pfu DNA Polymerase online at:
www.promega.com/citations/
*Not available in North America.
Tli DNA Polymerase
Cat.#: M7101 (50u; 40 reactions)
Supplied with enzyme (5u/l), Thermophilic DNA
Polymerase 10X Reaction Buffer and 25mM MgCl
2
.
Sufficient to give the indicated number of 50l
reactions using 1.25u of enzyme/reaction.
Citations for use of Tli DNA Polymerase online at:
www.promega.com/citations/
3
9
7
3
M
A
0
2
_
3
A
Pfu Tli Taq
0
1
2
3
4
5
6
7
8
A
c
c
u
r
a
c
y
1
0
5
DNA Polymerase
M 30 3 0.3 0.03 0.0
Promega
M 30 3 0.3 0.03 0.0
Supplier A
bp
1,500
1,000
500
100
1.2kb
product
2
3
5
5
T
A
0
8
_
8
A
A. B.
Proofreaders have a 3
5
exonuclease activity that is lacking
in
non-proofreading
enzymes like Taq
DNA Polymerase. Proofreading
enzymes
can degrade primers if the reaction is
allowed to sit for too long
prior to
amplification. We recommend setting
up
reactions on ice and adding
the
proofreading
polymerase just prior to
placing
the reaction into a preheated
thermal cycler.
U
sag
e
H
int: Inc
re
ase
am
plific
ation e
xte
nsion
tim
e
s 2
X
ove
r stand
ard
T
aq D
N
A
Polym
e
rase
(e.g
., 2
m
in/k
b)
and
ad
d
2
3
c
yc
le
s.
Promega DNA Analysis Notebook
22
Amplifying DNA
Hot Start Methodology
Hot start PCR is a commonly used technique to reduce
nonspecific amplification. One cause of nonspecific
amplification is the assembly of PCR reactions at room
temperature or on ice. Under these conditions, the PCR
primers may be able to anneal to various non-
complementary positions on the template. Although
activity of thermostable DNA polymerases at room
temperature or 4C is usually less than 25%, they can
extend nonspecifically annealed primers at these
temperatures. Any newly synthesized product is
completely complementary to the PCR primer, allowing
the primer to anneal specifically to this region during
PCR, resulting in an undesired amplification product.
Hot start PCR can also reduce the amount of primer-
dimer formed. Primer-dimers result from
complementarity between the 3 ends of the PCR primers.
At room temperature or on ice, these complementary
regions anneal and the polymerase extends the ends to
produce a primer-dimer. Primer-dimers often appear as a
diffuse band at ~50100bp on ethidium bromide-stained
gels. Both nonspecific products and primer-dimers can
compete with the desired amplification reaction for
reagents. By avoiding the conditions that lead to
nonspecific amplification, hot start PCR can improve the
yield of the desired PCR product.
There are several ways to perform hot start PCR. The
reaction can be assembled on ice or at room
temperature, omitting the DNA polymerase until the
reaction has been placed in the thermal cycler and
heated to 6065C. Once the reaction has reached
6065C the desired amount of polymerase can be
added. This prevents the polymerase from extending
primers until the higher temperature is reached and
primer annealing is more specific. The method is quite
effective but can be labor-intensive, particularly if
dozens of amplification reactions are involved.
Another approach to hot start PCR involves the use of
wax to physically sequester one or more critical reaction
components until the appropriate temperature is
reached. Wax beads can be added to a PCR before the
addition of the DNA polymerase. Heating the PCR to
60C melts the wax, which forms a liquid layer over the
surface of the reaction, eliminating the need for mineral
oil. Upon cooling to 4C, the wax solidifies. The DNA
polymerase is added onto this wax layer and, as the
PCR is heated during the first denaturation step, the wax
melts and the polymerase can access the other PCR
reagents. This method is labor intensiverequiring an
additional heating and cooling step to prepare the wax
layer. Also, opening the PCR tube to add the polymerase
increases the risk of contamination. Additionally, the
solid wax layer that forms upon cooling to 4C will clog
pipet tips when attempting to break through the wax to
pipet the PCR. Thus, it is often necessary to use one
pipet tip to puncture the wax layer and a second pipet
tip to remove the PCR products.
TaqBead Hot Start Polymerase
Enter Promegas TaqBead Hot Start Polymerase
(f)
. By
impregnating a wax bead with Taq DNA Polymerase, the
additional heating and cooling steps to form the wax
layer are eliminated. A single bead is added to each 50l
reaction, and as the reaction is heated, the wax melts
and releases the polymerase. The molten wax rises to
the surface of the PCR where it forms an incomplete
barrier. In thermal cyclers with heated lids, a hole
remains above the reaction to ease pipetting. To prevent
evaporation in thermal cyclers without heated lids, we
recommend adding mineral oil to each PCR. The molten
wax and mineral oil will mix during the thermal cycling
to form a single layer, which solidifies when the reaction
is cooled to 4C and can clog pipet tips.
TaqBead Hot Start Polymerase
Cat.#: M5661 (100 reactions)
Supplied with 100 beads (1.25u Taq DNA Polymerase
in Storage Buffer B/bead), Thermophilic DNA
Polymerase 10X Reaction Buffer, and 25mM MgCl
2
.
Protocol:
www.promega.com/tbs/tb247/tb247.html
Citations for use of TaqBead Polymerase online at:
www.promega.com/citations/
Hot start amplification reduces the yield of nonspecific amplification products.
Aliquots of 10, 1 or 0.1pg pGEM
-luc Vector
(h,p)
(Cat.# E1541) were diluted in 30ng of
Human Genomic DNA (Cat.# G3041). A 1.8kb luciferase gene product was amplified by
PCR using Taq DNA Polymerase (Storage Buffer B) or TaqBead Hot Start Polymerase in
Promega Reaction Buffer supplemented with 2mM MgCl
2
. Details are provided in Miller, K.,
Smith, R. and Storts, D. (1996) Improved PCR amplification using TaqBead Hot Start
Polymerase. Promega Notes 60, 26.
bp
2,645
1,605
1,198
676
517
396
primer-
dimer
1.8kb
M 10 1 0.1 10 1 0.1
Taq
Cold Start
TaqBead
Hot Start
pg of
target
1
5
8
6
T
A
0
9
_
6
A
www.promega.com techserv@promega.com
23
Amplifying DNA
dNTPs
Promega is a premier supplier of high-quality dNTPs in
bulk form or premixed in the PCR Nucleotide Mix.
Promegas dNTPs are >99% triphosphate with verified
concentrations. All dNTPs, whether bulk or premixed,
are DNase- and RNase-free, and are functionally tested
in amplification reactions. The PCR Nucleotide Mix is
also functionally tested in RT-PCR.
PCR Nucleotide Mix
Cat.#: C1141 (200l; 200 reactions)
C1145 (1,000l; 1,000 reactions)
The PCR Nucleotide Mix supplies a single solution
containing each dNTP (dATP, dTTP, dGTP, dCTP) at
10mM. Reaction size is considered to be 200M of
each dNTP in a 50l reaction. Each reaction uses 1l
of PCR Nucleotide Mix.
Custom and bulk PCR Nucleotide Mix sizes are
available.
Protocol:
www.promega.com/tbs/9pic114/9pic114.html
Set of dATP, dCTP, dGTP and dTTP
Cat.#: U1330 (10mol each; 1,000 reactions)
U1240 (40mol each; 4,000 reactions)
U1410 (200mol each; 20,000 reactions)
Set of dUTP, dATP, dCTP and dGTP
Cat.#: U1335 (10mol each; 1,000 reactions)
U1245 (40mol each; 4,000 reactions)
Each dNTP is supplied at 100mM. Reaction size is
considered to be 200M each dNTP in a 50l PCR
reaction.
Custom and bulk dNTP sizes are available.
RT-PCR functional assay using PCR Nucleotide Mix. The dNTPs were used following
50 freeze-thaw cycles. Amounts of template RNA: lane 1, 25fmol; lane 2, 2.5fmol; lane 3,
250amol; lane 4, 25amol; lane 5, 2.5amol; lane 6, 250zmol; lane 7, no template control.
Stability of dNTPs. The integrity of the RT-PCR products on the gel demonstrates the
stability of the dNTPs after storage under the conditions listed. The dNTPs used in each RT-
PCR were stored as follows prior to use: Lane 2, 1 freeze-thaw cycle; lane 3, 50 freeze-thaw
cycles; lane 4, 1 year at 20C; lane 5, negative PCR control; lanes 1 and 6, 100bp DNA
Ladder (Cat.# G2101).
1 2 3 4 5 6
3
5
2
7
T
A
0
8
_
1
A
bp
1,500
1,000
500
1 2 3 4 5 6 7
3
5
2
8
T
A
0
8
_
1
A
Promega DNA Analysis Notebook
24
Amplifying DNA
Routine PCR
Product Size Cat.#
PCR Master Mix
(f,g)
100 reactions M7502
1,000 reactions M7505
For Laboratory Use. A reaction consists of 25l of the 2X PCR Master Mix in a 50l total volume. Supplied with Nuclease-Free Water.
Product Size Cat.#
GoTaq DNA Polymerase
(e,g)
100u M3001
500u M3005
2,500u M3008
For Laboratory Use. Supplied with 5X Green GoTaq Reaction Buffer and 5X Colorless GoTaq Reaction Buffer. Both buffers contain 1.5mM Mg
2+
at the 1X concentration.
Product Size Cat.#
PCR Core System I
(f)
200 reactions M7660
PCR Core System II
(f)
200 reactions M7665
For Laboratory Use. PCR Core Systems provide Taq DNA Polymerase in Storage Buffer B, PCR Nucleotide Mix, Taq DNA Polymerase 10X Reaction Buffers with and without MgCl
2
, and 25mM MgCl
2
sufficient for 200 50l reactions containing 1.25u of Taq DNA Polymerase. PCR Core System II also contains Positive Control Plasmid DNA template, and Upstream and Downstream Control
Primers.
Product Size Cat.#
Taq DNA Polymerase in Storage Buffer A
(e)
100u M1861
(Supplied with Taq DNA Polymerase 10X Reaction Buffer without MgCl
2
, and 25mM MgCl
2
.) 500u M1865
2,500u M1868
Taq DNA Polymerase in Storage Buffer A
(e)
100u M2861
(Supplied with Taq DNA Polymerase 10X Reaction Buffer with MgCl
2
, giving 1.5mM Mg
2+
at the 1X concentration.) 500u M2865
2,500u M2868
Taq DNA Polymerase in Storage Buffer B
(e)
100u M1661
(Supplied with Taq DNA Polymerase 10X Reaction Buffer without MgCl
2
, and 25mM MgCl
2
Solution.) 500u M1665
2,500u M1668
Taq DNA Polymerase in Storage Buffer B
(e)
100u M2661
(Supplied with Taq DNA Polymerase 10X Reaction Buffer with MgCl
2
, giving 1.5mM Mg
2+
at the 1X concentration.) 500u M2665
2,500u M2668
For Laboratory Use.
Proofreading Polymerases
Product Size Cat.#
Pfu DNA Polymerase
(e)
* 100u M7741
(Supplied with Pfu DNA Polymerase 10X Reaction Buffer with MgSO
4
, giving 2mM Mg
2+
at the 1X concentration.) 500u M7745
Tli DNA Polymerase
(e)**
(Supplied with Thermophilic DNA Polymerase 10X Reaction Buffer and 25mM MgCl
2
.) 50u M7101
*Not Available in North America. **For Laboratory Use.
www.promega.com techserv@promega.com
25
Amplifying DNA
Hot Start Polymerase
Product Size Cat.#
TaqBead Hot Start Polymerase
(f)
(Supplied with Thermophilic DNA Polymerase 10X Reaction Buffer and 25mM MgCl
2
.) 100 reactions M5661
For Laboratory Use. One bead per reaction; 1.25u Taq DNA Polymerase per bead.
dNTPs
Product Size Cat.#
PCR Nucleotide Mix 200l C1141
(Contains 10mM each dNTP; use 1l per 50l reaction.) 1,000l C1145
Set of dATP, dCTP, dGTP, and dTTP 10mol U1330
(100mM each dNTP. Individual tubes available.) 40mol U1240
200mol U1410
Set of dUTP, dCTP, dGTP, and dATP
(q)
10mol U1335
(100mM each dNTP.) 40mol U1245
For Laboratory Use.
Accessories
Product Size Cat.#
Promega 10 Barrier Tips, 960/pk 0.510l A1491
Promega 10E Barrier Tips, 960/pk 0.510l A1501
Promega 10F Barrier Tips, 960/pk 0.510l A1511
Promega 20 Barrier Tips, 960/pk 220l A1521
Promega 100 Barrier Tips, 960/pk 10100l A1541
Promega 200 Barrier Tips, 960/pk 50200l A1551
Promega 1000 Barrier Tips, 480/pk 1001,000l A1561
Mineral Oil* 12ml DY1151
Nuclease-Free Water* 150ml P1195
*For Laboratory Use.
Tip and Pipette Compatibility Guide
Oxford
Size Pipetman
Eppendorf
Benchmate
Finnpipette
SV
Gel & PCR
Clean-Up System
Wizard
PCR
Preps DNA
Purification
System
Wizard
SV 96
PCR Clean-Up
System
Wizard
MagneSil PCR
Clean-Up System
Direct Purification
www.promega.com techserv@promega.com
27
PCR Clean-Up
Wizard
SV Gel and PCR Clean-Up System. DNA fragments of the sizes indicated
were analyzed before (U) and after (P) direct purification from amplification reactions.
Elution volume versus recovery for a 700bp PCR product purified directly from
an amplification reaction using the Wizard
X-100 92%
0.1% Tween
20 87%
0.1% NP-40 82%
5% Glycerol 87%
5% Formamide 90%
5% DMSO 87%
0.5M Tetramethylene Sulfoxide 94%
0.4M Sulfolane 94%
0.4M 2-Pyrolidene 95%
1mM Tartrazine 100%
1% Ficoll
-400 100%
3
9
7
2
M
A
0
2
_
3
A
10 15 25 50 75 100
0
10
20
30
40
50
60
70
80
90
100
P
e
r
c
e
n
t
R
e
c
o
v
e
r
y
Elution Volume (l)
L
i
n
e
a
r
D
N
A
a
s
b
i
g
a
s
1
0
k
b
c
a
n
b
e
p
u
r
if
ie
d
w
i
t
h
u
p
t
o
9
5
%
r
e
c
o
v
e
r
y
.
Promega DNA Analysis Notebook
28
PCR Clean-Up
Wizard
SV 96
PCR Clean-Up System
The Wizard
2000 and
FX instruments.
Microarray of purified PCR products. Representative microarray blocks of PCR
product purified using the Wizard
2000 liquid
handler. PCR fragments of 100, 200, 300, 500 and 1,000bp were purified using the
Wizard
l
l
w
o
r
k
i
n
S
V
9
6
.
M
a
n
u
a
l
o
r
a
u
t
o
m
a
t
e
d
9
6
-
w
e
ll
P
C
R
p
u
r
if
ic
a
t
io
n
.
www.promega.com techserv@promega.com
29
PCR Clean-Up
Wizard
MagneSil
PCR Clean-Up System
The Wizard
384 Magnetic
Separation Device is also available (Cat.# V8241).
The MagneSil PCR Clean-Up procedure is fast and
reliable. PCR products bind to MagneSil particles in
the presence of guanidine hydrochloride and remain
tightly bound during washing. Purified DNA is eluted in
water and may be used for automated fluorescent
sequencing and microarray spotting. The MagneSil
PCR Clean-Up System is ideally suited for reactions
prepared using PCR Master Mix and GoTaq DNA
Polymerase. It also works well with reactions prepared
using AmpliTaq
DNA Polymerase.
Array images after hybridization with Cy
3 and Cy
5 fluorescent control
E. coli targets. E. coli genomic DNA was amplified using 96 unique primer pairs. The
Wizard
3- or Cy
MagneSil PCR
Clean-Up System. PCR amplimers were purified from standard amplification reactions
performed using either PCR Master Mix or AmpliTaq
DNA Polymerase.
Wizard
DNA
Polymerase
3
5
7
8
T
A
1
1
_
1
A
E. coli Control Array
Cy
3 Cy
5
Quality tested for
success in fluorescent
BigDye
Sequencing
with >98% accuracy
over 600 bases read.
Promega DNA Analysis Notebook
30
PCR Clean-Up
Wizard
PCR Preps
DNA Purification System
The Wizard
PCR Preps System. Reactions are shown before (lanes 2 and 4) and after
(lanes 3 and 5) purification.
1,000
bp
M M
500
200
50
Before Purification After Purification
0
9
3
5
T
A
1
1
_
4
A
1 2 3 4 5
0
3
4
8
T
A
0
8
_
3
A
Wizard
SV 96 PCR Clean-Up System provides a manual or automated system for 96-well direct PCR purification by vacuum. Compatible with a wide variety of PCR
additives and all Promega amplification reaction buffers.
Product Size Cat.#
Wizard
MagneSil PCR Clean-Up System provides an automated 96- or 384-well system for direct purification of PCR products. Compatible with standard reactions
using Promega's PCR Master Mix or GoTaq DNA Polymerase.
Product Size Cat.#
Wizard
PCR Preps DNA Purification System is a manual, resin-based vacuum system for direct purification or isolation from agarose gels.
Related Products
Product Size Cat.#
Agarose, LE, Analytical Grade 100g V3121
500g V3125
Agarose, Low Melting Point, Analytical Grade 25g V2111
Blue/Orange Loading Dye, 6X* 3ml (3 1ml) G1881
TAE Buffer, 10X 1,000ml V4271
TAE Buffer, 40X 1,000ml V4281
TBE Buffer, 10X 1,000ml V4251
*For Laboratory Use.
Promega DNA Analysis Notebook
32
Cloning PCR DNA
Overview
Cloning PCR products into plasmid vectors is a
common downstream application of PCR. When PCR
was in its infancy, researchers found that it was not easy
to clone PCR products by simple blunt-end ligation into
blunt-ended plasmid vectors because some
thermostable DNA polymerases, including Taq DNA
Polymerase, add a single nucleotide base extension to
the 3-end of blunt DNA in a template-independent
fashion (1,2). Most commonly the base added is
adenine, leaving what is called an A overhang. To
overcome this, researchers had to treat PCR products to
blunt-end the PCR fragments prior to cloning. Such
experiments often suffered from low cloning efficiencies.
Another commonly used strategy for PCR cloning is to
add restriction enzyme recognition sites to the ends of
PCR primers (3). The PCR product is then digested and
cloned into the desired vector. When using this method,
care must be exercised in primer design because not all
enzymes cleave efficiently at the ends of DNA fragments,
and you may not be able to use every enzyme you
desire (4,5). Some enzymes require extra bases outside
the restriction enzyme recognition site, adding further
expense to the PCR primers as well as increasing the
risk of annealing to unrelated sequences in the genome.
The fact that amplicons generated with Taq DNA
Polymerase typically have A overhangs led to the
method referred to as T-vector cloning. In essence, the
plasmid cloning vector is engineered to contain 3-T
overhangs that match the 3-A overhang of the amplicon
(6). The A-tailed amplicon is directly ligated to the T-
tailed plasmid vector, and there is no need for further
enzymatic treatment of the amplicon other than the
action of T4 DNA Ligase. Promega has systems based
on this technology for routine subcloning, direct
mammalian expression and bacterial expression.
References
1. Clark, J.M. (1988) Novel non-template nucleotide addition reactions
catalyzed by procaryotic and eucaryotic DNA polymerases. Nucl. Acids Res.
16, 967786.
2. Mole, S.E., Iggo, R.D. and Lane, D.P. (1989) Using the polymerase chain
reaction to modify expression plasmids for epitope mapping. Nucl. Acids
Res. 17, 3319.
3. Scharf, S.J., Horn, G.T. and Erlich, H.A. (1986) Direct cloning and sequence
analysis of enzymatically amplified genomic sequences. Science 233,
107678.
4. Kaufman, D.L. and Evans, G.A. (1990) Restriction endonuclease cleavage at
the termini of PCR products. BioTechniques 9, 3046.
5. Digestion of restriction sites close to the end of linear DNA. In: Restriction
Enzyme Resource Guide. Promega Corporation.
www.promega.com/guides/re_guide/chaptwo/2_6.htm
6. Mezei, L.M. and Storts, D.R. (1994) Cloning PCR Products. In: PCR
Technology Current Innovations. Griffin, H.G. and Griffin, A.M. (eds). CRC
Press, pp. 217.
pGEM
-T &
pGEM
-T Easy
Vectors
pTARGET Mammalian
Expression Vector
PinPoint Xa-1
T-Vector
T
A
A
T
T
T
T
T
For cloning PCR products, the choice is yours
3
7
7
9
M
A
0
7
_
2
A
Basic
Subcloning
Direct Mammalian
Expression
Direct Bacterial
Expression
www.promega.com techserv@promega.com
33
Cloning PCR DNA
Basic Subcloning
pGEM
-T and pGEM
-T and pGEM
-T Easy Vector
Systems
(h,i)
were designed for just that purpose. The
vectors are based on the pGEM
-5Zf(+) Vector
(h)
backbone.
The pGEM
-T
and -T Easy Vectors are provided with 2X Rapid Ligation
Buffer, which allows efficient ligation in just 1 hour with the
supplied T4 DNA Ligase. You can either supply your own
favorite E. coli competent cells or purchase the system with
Promegas high-efficiency JM109 Competent Cells. The
choice is yours.
pGEM
-T Vector System I
(you supply the competent cells)
Cat.#: A3600 (20 reactions)
pGEM
-T Vector System II
(supplied with High Efficiency JM109 Competent Cells)
Cat.#: A3610 (20 reactions)
Protocol:
www.promega.com/tbs/tm042/tm042.html
Citations for use of the pGEM
Xmn I 1994
Nae I
2692
lacZ
f1 ori
1 start
14
20
26
31
37
46
55
62
62
73
75
82
94
103
112
126
SP6
T T
pGEM
-T
Vector
(3000bp)
0
3
5
6
V
A
0
4
_
3
A
Select recombinants by
blue/white selection.
Drop out your
insert with a
single Bst Z I
digest.
S
e
q
u
e
n
c
e
in
s
e
r
t
s
w
it
h
:
S
P
6
P
r
o
m
o
t
e
r
P
r
im
e
r
T
7
P
r
o
m
o
t
e
r
P
r
im
e
r
p
U
C
/
M
1
3
F
o
r
w
a
r
d
P
r
im
e
r
p
U
C
/
M
1
3
R
e
v
e
r
s
e
P
r
im
e
r
Promega DNA Analysis Notebook
34
Cloning PCR DNA
For maximum subcloning efficiency, purify the PCR
product before subcloning. The presence of PCR
primers and primer-dimers can reduce subcloning
efficiency. See Chapter 3 for more information.
If you do not purify your PCR product, at least make
the amplification as specific as possible. The cleaner
the product, the better the ligation efficiency. Try to
avoid production of primer-dimers by optimizing the
amplification reaction conditions. See Chapter 2 for
more information on optimizing PCR.
Example of Transformation Efficiency Calculation:
After 100l competent cells are transformed with
0.1ng uncut plasmid DNA, the transformation
reaction is added to 900l of SOC medium
(0.1ng DNA/ml). A 1:10 dilution with SOC medium
(0.01ng DNA/ml) is made, and 100l is plated on
each of two plates (0.001ng DNA/100l). If 200
colonies are obtained (average of two plates), what is
the transformation efficiency?
200cfu
1ng
= 2 10
8
cfu/g DNA
0.001ng 10
3
g
pGEM
-T Easy System
online at:
www.promega.com/citations/
Relationship between incubation time and cloning efficiency using the 2X
Rapid Ligation Buffer and the pGEM
-T Easy Vector was ligated into the vector using a 1:1 vector:insert
molar ratio. The Rapid Ligation Buffer and T4 DNA Ligase were used in ligation reactions,
which were set up at room temperature (24C) and allowed to proceed from 0.25 to 16
hours. Number of white colonies (transformants) versus time of ligation are shown. This
graph was adapted from Table 2 in Frackman, S. and Kephart, D. (1999) Rapid Ligation for
the pGEM
-T and pGEM
-T Easy
Vector
(3015bp)
Apa I
Aat II
Sph I
BstZ I
Nco I
BstZ I
Not I
Sac II
EcoR I
Spe I
EcoR I
Not I
BstZ I
Pst I
Sal I
Nde I
Sac I
Bst X I
Nsi I
T7
Xmn I 2009
Nae I 2707
lacZ
f1 ori
1
start
14
20
26
31
37
43
43
49
64
70
77
77
88
90
97
109
118
127
141
SP6
T T
1
4
7
3
V
A
0
5
_
6
A
Drop out your
insert with a
single Bst Z I,
EcoR I or
Not I digest.
Sequence inserts with:
SP6 Promoter Primer
T7 Promoter Primer
pUC/M13 Forward Primer
pUC/M13 Reverse Primer
For maximum efficiency,
use competent cells
capable of at least
1 x 10
8
cfu/g DNA.
4
0
1
9
M
A
0
3
_
3
A 0
50
100
150
200
250
300
350
400
450
0 4 8 10 12 14 16
N
u
m
b
e
r
o
f
W
h
i
t
e
C
o
l
o
n
i
e
s
Hours
2 6
www.promega.com techserv@promega.com
35
Cloning PCR DNA
Direct Mammalian Expression
pTARGET Mammalian Expression Vector System
The pTARGET Mammalian Expression Vector
(i,j)
is
designed to streamline your experiments, allowing you
to go from PCR and T-vector cloning directly to
expression analysis in a mammalian system. The
pTARGET Vector is based on the popular pCI-neo
Vector
(h,j)
(Cat.# E1841) and delivers powerful
mammalian expression through the CMV promoter. The
vector also has the neomycin resistance necessary for
G-418 Sulfate selection of stable transformants.
The pTARGET Vector is the only mammalian
expression T-vector offering blue/white selection of
recombinants. The vector contains the lacZ -peptide
fragment to complement the -fragment of lacZ that is
expressed in common E. coli strains like JM109,
DH5 and XL-1 Blue. See page 33 for more
explanation of blue/white selection.
pTARGET Mammalian Expression Vector System
Cat.#: A1410 (20 reactions and 20 transformations
with high-efficiency JM109 Competent Cells)
Protocol:
www.promega.com/tbs/tm044/tm044.html
Citations for use of the pTARGET System online at:
www.promega.com/citations/
The pTARGET Vector contains the simian virus 40
(SV40) enhancer and early promoter region
upstream of the neomycin phosphotransferase gene.
The SV40 early promoter contains the SV40 origin of
replication, which will induce transient, episomal
replication of the pTARGET Vector in cells expressing
the SV40 large T antigen such as COS-1 or COS-7
cells (1). Consequently, the copy number of the
vector will increase in these SV40-transformed cell
lines and give higher transient expression than in
other cell types.
1. Gluzman, Y. (1981) SV40-transformed simian cells support the
replication of early SV40 mutants. Cell 23, 17582.
pTARGET Mammalian Expression Vector has been
used for transient expression in many cell lines
including:
COS-1 SV40-transformed monkey kidney
COS-7 SV40-transformed monkey kidney
H9c2 rat myoblast
Primary human melanoma
The pTARGET Mammalian Expression Vector has
been used to create stable transfectants by G-418
Sulfate selection in many cell lines including:
1376 TCC human bladder transitional cell carcinoma
293 human embryonic kidney cell
A549 human adenocarcinoma
CHO Chinese hamster ovary
NIH/3T3 mouse fibroblast
J82 human bladder transitional cell carcinoma
PS120 Chinese hamster lung fibroblast
RAW264.7 mouse monocyte/macrophage cell line
T24 human bladder transitional cell carcinoma
U937 human leukemic cells
See Promegas online citation database for further
examples and details: www.promega.com/citations/
Sgf I 664
I-Ppo I
851
Bgl II 5665
SV40 Enhancer/
EarlyPromoter
SV40 Late
poly (A)
fl ori Synthetic
poly(A)
Amp
r
ori
CMV
Enhancer/Promoter
Intron
Neo
pTARGET
Vector
(5670bp)
T
T
EcoR I
BamH I
Nhe I
Xho I
Mlu I
lacZ
lacZ
Sma I
Kpn I
Sal I
Acc I
Not I
EcoR I
T7
1250
1256
1264
1270
1276
1293
1301
1303
1304
1311
1318
T overhangs
1
5
0
5
V
A
0
7
_
6
A
S
equence inserts w
ith:
T
7
Prom
oter Prim
er
pT
ARG
ET
S
equencing
Prim
er
T
h
e
only
m
am
m
alian
e
xpre
ssion
T
-
v
e
c
tor
c
apable
of
blue
/w
h
ite
sc
re
e
nin
g
.
Drop out your insert
with a single
Eco R I digest.
Promega DNA Analysis Notebook
36
Cloning PCR DNA
Direct Mammalian Expression (continued)
pTARGET Mammalian Expression Vector System
Expression of various reporter proteins using either the pTARGET Mammalian Expression Vector or the pCI-neo Mammalian Expression Vector. The pTARGET Vector
was designed from the pCI-neo Vector (Cat.# E1841). Vector sequences for blue/white selection do not interfere with expression. T.U. = Turner light units. Details of this experiment may be
found in Brondyk, B. (1996) pTARGET Vector: A new mammalian expression T-Vector. Promega Notes 58, 27.
Need transfection grade plasmid DNA?
Promega has the system.
Wizard MagneSil Tfx System
For automated 96-well transfection-grade plasmid DNA
purification.
Cat.#: A2380 (4 96 preps)
A2381 (8 96 preps)
Protocol:
www.promega.com/tbs/tb314/tb314.html
For information on automated methods visit:
www.promega.com/automethods/
For expression in mammalian systems, your amplicon
should contain an initiation AUG codon and a stop
codon. Ideally the AUG codon is in the context of a
Kozak consensus sequence: (A or G)CCAUGG (1). Be
sure the initiation codon is the first AUG codon
encountered in the sequence.
1. Kozak, M. (1987) At least six nucleotides preceding the AUG initiator
codon enhance translation in mammalian cells. J. Mol. Biol. 196, 94750.
20
40
60
80
100
120
140
0
H
u
m
a
n
G
r
o
w
t
h
H
o
r
m
o
n
e
A
c
t
i
v
i
t
y
(
n
g
/
m
l
c
o
n
d
.
m
e
d
i
u
m
)
F
i
r
e
f
l
y
L
u
c
i
f
e
r
a
s
e
A
c
t
i
v
i
t
y
(
T
.
U
.
/
p
l
a
t
e
1
0
6
)
Cells Transfected
C
A
T
A
c
t
i
v
i
t
y
(
C
P
M
/
p
l
a
t
e
1
0
6
)
-
g
a
l
a
c
t
o
s
i
d
a
s
e
A
c
t
i
v
i
t
y
(
u
n
i
t
s
/
p
l
a
t
e
1
0
-
3
)
1
2
3
4
5
6
7
8
0
pCI-neo
pTARGET
pCI-neo
pTARGET
pCI-neo
pTARGET
pCI-neo
pTARGET
pCI-neo
pTARGET
R
e
n
i
l
l
a
L
u
c
i
f
e
r
a
s
e
A
c
t
i
v
i
t
y
(
T
.
U
.
/
p
l
a
t
e
1
0
6
)
HeLa NIH3T3
Cells Transfected
HeLa NIH3T3
Cells Transfected
HeLa NIH3T3
Cells Transfected
HeLa NIH3T3
Cells Transfected
HeLa NIH3T3
2.5
2
3.5
3
1.5
0.5
1
0
10
20
30
40
50
60
0
0
5
10
15
20
25
30
35
40
A. C. B.
D. E.
1
5
3
9
M
A
0
7
_
6
A
Learn about transfection and tools available from
Promega in the Transfection Guide available online at:
www.promega.com/guides/, or request literature
#BR041 from your local Promega representative.
Experimental details
available at:
www.promega.com/pnotes/
58/5189a/5189a.html
www.promega.com techserv@promega.com
37
Cloning PCR DNA
Direct Bacterial Expression
PinPoint Xa-1 T-Vector Systems
The PinPoint Xa-1 T-Vector
(h,i,t)
provides a convenient
means to clone and express PCR products in E. coli.
Fusion proteins made using the PinPoint Xa-1
T-Vector are easily expressed and purified. The
PinPoint Xa-1 T-Vector carries a segment encoding
a polypeptide that becomes biotinylated in E. coli and
subsequently functions as a purification tag (1).
Biotinylated fusion proteins produced with this system
can be affinity-purified using the SoftLink Soft Release
Avidin Resin
(u,v)
(Cat.# V2011). This proprietary resin
allows elution of the fusion protein under nondenaturing
conditions. The TetraLink Tetrameric Avidin Resin
(w)
(Cat.# V2591) also can be used to capture biotinylated
proteins irreversibly as a way to generate affinity resins.
Reference
1. Cronan, J.E., Jr. (1990) Biotination of proteins in vivo. A post-translational
modification to label, purify, and study proteins. J. Biol. Chem. 265,
1032733.
Protein expression using the PinPoint Xa-1 T-Vector System. Use with
SoftLink Soft Release Avidin Resin (Cat.# V2011) to purify fusion proteins. For details,
see the PinPoint Xa-1 T-Vector System Technical Bulletin #TB234.
PinPoint Xa-1 T-Vector System I
(you supply the competent cells)
Cat.#: V2610 (20 reactions)
Pinpoint Xa-1 T-Vector System II
(supplied with high-efficiency JM109 Competent Cells)
Cat.#: V2850 (20 reactions and 20 transformations
with JM109 Competent Cells)
Protocol:
www.promega.com/tbs/tb234/tb234.html
Citations for use of the PinPoint Xa-1 T-Vector
System online at:
www.promega.com/citations/
Note: Amplification primers must be designed to clone
in frame with the PinPoint Fusion Protein. Full
details are provided in Technical Bulletin #TB234.
389
392
400
404
410
414
422
430
435
452
Sal I 52
BstX I 264
Nde I
532
Factor
Xa site
379-390
Xmn I 1106
Sca I 1225
Pvu I 1337
Fsp I 1483
ori
Sac I
377
Hpa I 2777
T7 promoter Cla I 3018
tac promoter
EcoR I 3277
Pst I 3296
SP6
Nru I
Hind III
Pvu II
BamH I
Acc65 I
Kpn I
Bgl II
Sma I
Not I
purification
tag sequence
1-386
Amp
r
PinPoint
Xa-1
T-Vector
(3331bp)
1
1
1
8
V
A
0
7
_
5
A
1. Wash.
2. Elute with free biotin.
PinPoint
Expression Vector
1. Express fusion protein in E. coli.
2. Lyse cells, digest with nuclease.
3. Pellet cell debris.
Biotinylated
fusion protein
in crude cell
supernatant
Detection using
avidin reagents
Proteolytic removal of
biotinylated tag peptide
0
3
6
7
M
C
0
3
_
3
A
Bind to SoftLink Soft Release
Avidin Resin.
Sequence inserts with:
SP6 Promoter Primer
PinPoint Vector
Sequencing Primer
Promega DNA Analysis Notebook
38
Cloning PCR DNA
PCR Cloning Techniques
Rapid PCR-Based Screen for Orientation of Insert
All Promega PCR cloning vectors have some unique
landmarks, including RNA promoter primer binding sites
allowing easy sequencing of inserts. The primer binding
sites can also be used to rapidly screen for insert
orientation. For example, we cloned a 1.8kb insert into
the pGEM
-T and pGEM
-T
Easy Vector Systems Technical Manual #TM042.
Ends Left by Various Thermostable Polymerases.
Taq DNA Polymerase 3A overhang*
GoTaq DNA Polymerase 3A overhang*
Tfl DNA Polymerase 3A overhang*
Tth DNA Polymerase 3A overhang*
Pfu DNA Polymerase Blunt
Tli DNA Polymerase Blunt
Long PCR mixes Blunt
Proofreading Polymerases Blunt
* All bases may be found at 3 overhang. A tends to occur most often.
T7 Orientation
Forward Primer
Reverse Primer SP6 Primer
T7 Primer
T7 1.8kb fragment SP6
SP6 Orientation
Reverse Primer
Forward Primer SP6 Primer
T7 Primer
T7 1.8kb fragment SP6
2
5
7
4
M
B
0
2
_
9
A
Start with 17l of purified PCR fragment generated by a
proofreading polymerase (e.g., Pfu DNA Polymerase).
Add 1l Taq DNA Polymerase 10X Reaction Buffer with MgCl
2
.
Add dATP to a final concentration of 0.2mM.
Add 5 units of Taq DNA Polymerase.
Add deionized water to a final reaction volume of 10l.
Incubate at 70C for 1530 minutes.
Use 12l in a ligation reaction with
Promegas pGEM
-T and pGEM
-T Easy Vector.
2
3
5
7
M
A
0
2
_
9
A
An A-tailing procedure for blunt-ended PCR fragments.
Colony PCR. Colonies were suspended in 50l sterile water, boiled for 10 minutes,
centrifuged at 16,000 g for 5 minutes, and 5l of the supernatant was used in each
amplification. The DNA was amplified by PCR in 50l volumes with 50pmol of each primer
and 1.25 units of Promegas Taq DNA Polymerase (Cat.# M1661). After an initial
denaturation of 2 minutes at 94C, the amplification profile was 35 cycles of denaturation
(94C for 30 seconds), annealing (55C for 1 minute) and extension (72C for 2.5
minutes); PCR was concluded with 1 cycle of 72C for 10 minutes. Amplification products
(8l) were analyzed on a 1% agarose gel containing ethidium bromide.
M 1 2 3 4
2
5
7
5
T
A
0
2
_
9
A
T7 + For.
bp
2,645
1,605
1,198
676
517
222
T7 + Rev.
1 2 3 4
For more information and techniques
for cloning PCR DNA, check out
Promega's Frequently Asked Questions
on the T-vector cloning systems at:
www.promega.com/faq/
www.promega.com techserv@promega.com
39
Cloning PCR DNA
PCRCloning Techniques (continued)
What PCR Cloning Controls Can Do For You
Each Promega PCR cloning system is provided with a
Control Insert. The ligation and subsequent transformation
of the Control Insert can give you a lot of information
about your ligation and transformation reactions.
The total number of blue colonies in Control Insert and
no-insert controls should be approximately equal. The
negative control may have some white colonies.
Bacterial Plates for Blue/White Selection
LB medium (per liter)
10g Bacto
-tryptone
5g Bacto
-yeast extract
5g NaCl
Adjust pH to 7.0 with NaOH.
Ampicillin Stock Solution
Dissolve at 50mg/ml in water. Filter sterilize. Store in
aliquots at 20C
IPTG stock solution (0.1M)
1.2g IPTG (Cat.# V3951)
Add water to 50ml final volume. Filter-sterilize and
store at 4C.
X-Gal (2ml)
100mg X-Gal (Cat.# V3941)
Dissolve in 2ml N,N-dimethyl-formamide. Cover with
aluminum foil and store at 20C.
LB plates with ampicillin/IPTG/X-Gal
Add 15g agar to 1 liter of LB medium. Autoclave.
Allow the medium to cool to 50C before adding
ampicillin to a final concentration of 100g/ml, then
supplement with 0.5mM IPTG and 80g/ml X-Gal and
pour the plates. Pour 3035ml of medium into 85mm
petri dishes. Let the agar harden. Store at 4C for up
to 1 month or at room temperature for up to 1 week.
Interpreting Results
Results with the experimental insert look like those
with the Control Insert in terms of efficiency and %
white colonies.
Successful experiment. Greater than 80% of the white
colonies should contain inserts.
Results with the experimental insert and Control
Insert look like the negative control.
Ligation has failed. Avoid multiple freeze/thaws of the
ligation buffer. Ligase buffer contains ATP and could
be damaged by freeze-thaws. You may need to aliquot
the ligase buffer into useful portions for your
experimental needs.
Few/No colonies with experimental insert, Control
Insert or negative control.
Transformation has failed. Reassess the competent
cells with an intact, supercoiled plasmid and
determine the transformation efficiency. Use cells
>1 10
8
cfu/g to insure >100 colonies from the
Control Insert ligation.
Experimental insert gives more blue colonies than
the Control Insert or negative control and less white
colonies than the Control Insert.
In-frame insertion with no interruption of the
-fragment. Although the pGEM
-T Vector Control
Insert will produce recombinants that generate white
colonies, the insertion of other DNA fragments into the
lacZ coding sequence may not result in white colonies
unless the fragments disrupt the lacZ reading frame.
Although this tends to occur most frequently with PCR
products of 500bp or less, inserts of up to 2kb have
been reported to result in blue colonies. Moreover,
some insert DNAs can also give pale blue colonies or
bulls eye colonies that have a blue center and a
white perimeter. In one case, we found that a 1.8kb
insert produced white colonies when oriented in one
direction and bulls eye colonies when oriented in the
opposite direction (1).
1. Knoche, K. and Kephart, D. (1999) Cloning blunt-end Pfu DNA
polymerase-generated PCR fragments into pGEM
-T Vector Systems.
Promega Notes 71, 1013.
Typical PCR Cloning Results Using pGEM
-T
Easy Vector and JM109 Competent Cells
(1.5 10
8
cfu/ng DNA).
Efficiency % White
(cfu/ng DNA) Colonies
Control Insert 1,110 92%
Control Insert 1,125 92%
No insert 92 0%
No insert 109 0%
Ligations performed at room temperature for 1 hour.
Promega DNA Analysis Notebook
40
Cloning PCR DNA
Basic Subcloning
Product Size Cat.#
pGEM
-T Vector System I
(h,i)
20 reactions A3600
pGEM
-T Vector System II
(h,i)
20 reactions A3610
pGEM
-T and pGEM
-T Easy Vector Systems I do not include competent cells. With System II, competent cells are provided.
Direct Mammalian Expression
Product Size Cat.#
pTARGET Mammalian Expression Vector System
(i,j)
20 reactions A1410
Competent Cells provided.
Direct Bacterial Expression
Product Size Cat.#
PinPoint Xa-1 T-Vector System I
(h,i,t)
(you provide competent cells) 20 reactions V2610
PinPoint Xa-1 T-Vector System II
(h,i,t)
(includes competent cells) 20 reactions V2850
SoftLink Soft Release Avidin Resin
(u,v)
1ml V2011
5ml V2012
TetraLink Tetrameric Avidin Resin
(w)
1ml V2591
5ml V2592
Primers
Product Size Cat.#
T7 Promoter Primer (10g/ml)[5-d(TAATACGACTCACTATAGGG)-3] 2g Q5021
SP6 Promoter Primer (10g/ml) [5-d(TATTTAGGTGACACTATAG)-3] 2g Q5011
pUC/M13 Forward Primer (10g/ml) [5-d(CGCCAGGGTTTTCCCAGTCACGAC)-3] 2g Q5601
pUC/M13 Reverse Primer (10g/ml) [5-d(TCACACAGGAAACAGCTATGAC)-3] 2g Q5421
pTARGET Sequencing Primer (10g/ml) [5-d(TTACGCCAAGTTATTTAGGTGACA)-3] 2g Q4461
PinPoint Vector Sequencing Primer [5-d(CGTGACGCGGTGCAGGGCG)-3] 2g V4211
DNA Purification Systems
Product Size Cat.#
Wizard