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Farmaceutisk

 molekylärbiologi  med  bioinformatik  


januari  2018  
           
 

Laboratory  Manual  

Recombinant  DNA  technology  [Day  1-­‐4  +  some  further  work  during  


the  Bioinformatics  part  of  the  course]  
Overview  and  aim    
In this exercise you will perform a realistic experiment of molecular biology spanning over
several days. You will be given a sample of E. coli bacteria containing one of three plasmids
(plasmid number 1, 2 or 3). Each of these plasmids carries a (for you) unknown gene. In the
end of the course you will be expected to provide information on the gene carried by “your”
plasmid.  

Isolation  of  DNA  insert  sequence  from  plasmid  


There are several different methods for amplification and preparation of recombinant DNA.
The basic steps in almost all methods are: I) transformation of required DNA into host
bacteria, II) isolation of bacteria incorporated with required DNA, III) amplification/growth and
harvest of bacterial culture, IV) release of DNA from bacteria by lysis, V)
purification/separation of plasmid DNA from unwanted components, i.e. cellular extract, VI)
measurement of the DNA concentration.

In this lab you will start with the growing of plasmid-containing bacteria, followed by harvest
of the bacteria, plasmid purification and restriction digestion to obtain the gene fragment
carried by your plasmid.

Day  1  

1. Casting  of  agar  plates. 25 ml of pre-warmed agar, containing 100µg/ml


ampicillin, (prepared by supervisor) is gently transferred to a 100 mm bacterial growth
dish. Try to avoid air bubbles. Leave the dish on the bench to solidify. Plasmids
containing required DNA sequence are Ampicillin-resistant.
2. An  Executive  Summary.  Read lab manual for days 1 to 4, and then, in groups,
write (maximum one A4) an Executive Summary that shows how the results from
each lab day are dependent on the lab the day before. Leave your summaries to the
lab supervisor during step 3, the same day.
3. Growth  of  bacterial  colonies. Dip the inoculation stick into the glycerol stock
(50 % transformed E.Coli required DNA plasmid and 50 % glycerol/water (1:1 vol/vol))
(prepared by supervisor). Streak the glycerol stock on to the bacteria dish by zigzag
movement. See pattern description below (Fig. 1). Put the dish cover on to the dish
and incubate the dish at 37°C overnight to let single-cell transformed colonies grow.
Do not forget to mark your bacteria dish with name and laboratory group number.

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Day  2  

1.    Amplification  of  DNA,  overnight  culture. Pick a single bacteria colony


from the selective bacteria plate. Use a yellow tip for picking the colony. Place the tip,
with bacteria from the colony in 2 ml LB (growth medium), containing the selective
antibiotic Ampicillin (100 µg/ml). Grow bacteria at 37° C with vigorous shaking (about
200-250 rpm) 12-16 h.  

Figure 1. Streaking out glycerol stock


onto selective bacteria dish

1. 1. Start and by zigzag streak


movement go down.
2. Start the new zigzag pattern
by streaking the inoculation
stick over the end part of
movement pattern 1. Do the
zigzag pattern across the
  dish (90 degree angle from
zigzag pattern 1).
   
3. Start the new zigzag pattern
by moving the inoculation
stick over the end part of
movement pattern 2. Do the
zigzag pattern in a 90

 
3.
degree angle from zigzag
pattern 2.

2.

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2.    Pipetting.    Correct and careful handling of the automatic pipettes is absolutely
essential for the following lab experiments. This exercise is designed to make you
acquainted with this equipment, helping you to get better results on Lab days 3 and 4.  
* Check out the pipettes. There are three different sizes, each for a specific range of
volumes. Each pipette has its own disposable tips (with the same color as that shown
on the pipette), which fits to the pipette and is attached when pipetting. The
disposable tip is the only part that should be in contact with the liquid you are
pipetting! Practice how to put on the tips and setting and reading different volumes.
Ask one of the supervisors if you feel unsure.
* Collect a tissue paper from one of the supervisors. Pipette water in volumes of 10 µl
(microliters), 100 µl and 500 µl to the paper as instructed by the supervisor. Show
your results to supervisor.
Please keep in mind that the pipettes are expensive and easily damaged. Do not
expose them to bumps or shocks and please do not drop them on the floor!

Day  3

Isolation  of  plasmid  DNA  from  E.  Coli  by  TENS  Miniprep  protocol.
a) Pellet bacteria from 1.5 ml of culture, in a 2 ml
eppendorf tube, by spinning @ 12000 rpm for 30
sec.
b) Pour off supernatant, except for the last 15-20 µl of
media which you leave in the tube.
c) Vortex to re-suspend pellet.
d) Add 300 µl of TENS-solution. Vortex for 3-5 sec and
place tube on ice for 6 min. The solution will become
sticky.
e) Add 150 µl 3.0 M sodium acetate (pH 5.2) - invert
tube 10-16 times.
f) Spin the tube @ 12000 rpm for 5 min to pellet
bacteria debris. In the meantime, add 0.9 ml of ice-
cold pure 95% ethanol to a new clean 1.5 ml-tube.
g) Transfer supernatant, by pouring, to the ethanol-
”spin” = snurra containing tube. Avoid transferring the precipitate.
(centrifugera) h) Mix by vortexing 3-5 sec.
i) Spin the tube @ 12000rpm for 3 min. Discard the
supernatant. Be careful to not disturb the pellet!
”vortex” = skaka j) Add 1 ml of 70% ethanol. Spin for 10 sec. Pour off
the supernatant. Be careful to not disturb the
pellet!
k) Repeat step j) one more time.
l) Remove the 70 % ethanol and air dry the pellet.
m) Resuspend the DNA pellet in 40 µl MilliQ water.
n) Clearly label your samples with name and laboratory
group number and give to supervisor for
measurement of the DNA concentration.

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Day  4  
Digestion of plasmid with a single restriction enzyme.
• Calculate the volume of plasmid DNA required for 1 µg DNA. Table for calculation
below (table 1).
• Mix the correct volume of DNA with 2 µl reaction buffer and MilliQ water to a total
reaction volume of 19 µl. Mix solution by pipetting up and down about 5-7 times.
• Add 1 µl restriction enzyme (10 U/µl). Use the correct restriction enzyme for
your plasmid! Plasmid number 1 should be cut with Bam HI. Plasmid 2 and 3
should be cut with NotI.
• Incubate reaction 30 min at 37°C. Quench digestion by incubating reaction at
80°C for 5 min in heating block.
• Mix your digestion reaction with 2 µl of loading dye. Apply your sample on a 1 %
agarose gel (prepared by supervisor). Run gel in electrophoresis buffer. The
supervisor will photograph the gel.
• Check the gel for size of your DNA-fragment. Expected sizes of the gene
fragments carried by plasmid 1, 2 and 3 are 1604 bp, 2369 bp and 1656 bp
respectively.
• If the gene fragment obtained has the expected size, the next step is to perform
DNA sequence analysis. Such samples are usually sent for sequencing to a
specially equipped laboratory, which performs sequencing for a charge (i e the
Uppsala Genome center).
• The sequence of your gene fragment will be given to you later in the course
(sequencing laboratories usually takes a few days to deliver). During the
computer lab exercises in the Bioinformatics part of the course you will work with
this sequence. Please include in your lab report the following information:
-which plasmid you worked with (no. 1, 2 or 3)
-the name of the recombinant gene carried by your plasmid
-the species from which the gene is derived
-a brief information of the function of this gene

DNA ____ µl (1µg)

Digestion buffer ____ µl

Restriction enzyme 1 µl

MilliQ water ____ µl (adjust to a total volume of 20 µl)

Table 1. Table  for  reaction(s)  

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Genotyping  by  PCR  [Day  5]  
Overview  and  aim    
In this exercise you will take part in a research project aimed to develop a transgenic “knock-
out” mouse. The gene of interest encodes a transport protein important for liver function.
Expression of this gene has also been discovered in the brain but its function there remains
unknown. Experiments using transgenic animals, where the normal gene for this protein has
been destroyed, could give information on the role of this transport protein in the brain.

When working with genetically modified animals it’s important to know what genotype the
animals have. For this, gene specific PCR analysis is required for every animal. When the
animals are weaned a biopsy is taken from the animal (tail or ear). DNA is then extracted
from the biopsy and PCR amplification is performed. The product is then analyzed on an
agarose gel.

In this part of the wet lab each laboratory group will get two DNA samples, obtained from the
ears of two different mice and you will perform a genotyping PCR for these animals. For this
specific genotyping there are specific PCR primer pairs for each of the gene variants: wild
type=WT and knock out=KO.

The animals from which your samples have been taken may be either:

-WILD TYPE (WT) meaning normal mice that have the normal unaltered gene

-KNOCK OUT (KO) meaning that they lack the gene of interest, instead they carry an
abnormal gene with no function (in an animal like this the gene has been successfully
“knocked out”)

-HETEROZYGOUS (HET) these mice have one copy of the normal gene and one copy of
the abnormal one

Remember to ALWAYS work on ice when performing PCR reactions.

You will get a pre-mix of the PCR reaction solution. This pre-mix contains nucleotides
(dNTP), buffer, MgCl2 and polymerase (for more information see Appendix). To this mix you
must add the correct PCR primers (forward and reverse) and the DNA from your samples.

1. First add 34 µl of the PCR premix to a 1.5 ml PCR tube. Add 2 µl of WT (wild type)-
specific forward primers. Then add 2 µl of WT (wild type)-specific reverse primers.
Divide this mixture between two new 0.2 ml tubes.

2. Then add another 34 µl of the PCR premix to a 1.5 ml PCR tube. Add 2 µl of KO
(knock out)-specific forward primers. Then add 2 µl of KO (knock out)-specific
reverse primers. Divide this mixture between two new 0.2 ml tubes.

3. You now have two tubes with PCR reaction solution containing WT-specific primer
pairs and two tubes containing KO-specific

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primer pairs

Always keep the solutions on ice


at all times!

4. Take 1 µl from one of your


DNA samples and add this
to one of the WT PCR-
reaction tubes.
5. Take 1 µl from one of your
DNA samples and add this
to one of the KO PCR-
reaction tubes.
6. Take 1 µl from the other
DNA sample and add this
to one of the WT PCR-
reaction tubes.
7. Take 1 µl from the same
DNA sample as in 6. and
add this to one of the KO
PCR-reaction tubes.

You will now have 4 PCR-reaction tubes (2 with WT-specific primers and 2 with KO-specific
primers).

When done leave the samples, ON ICE, for the supervisor who will run the PCR reaction. For
PCR program, see below. All samples will be run at the same time in the PCR. Do not forget
to mark your sample tubes according to which primers they contain (WT-specific or KO-
specific) and sample number (WT1/KO1 and WT2/KO2 for example) and your identification
(group number). After the PCR run you will identify your samples and add loading dye. Mix
your PCR reaction with 2 µl of loading dye. Load them on to an agarose gel in a specific way
and order, as instructed by supervisor. The supervisor will take a gel picture when the
electrophoresis if finished.

PCR program
Tid
(min/sek) °C
2 min 95
 
30 sek 95
40 sek 62 X  30  cycles     Fragment size:
40 sek 72 approx 200bp
6 min 72 50 or 100bp ladder
2 min 20 1 % agarose gel
130V
20-40 min
How to interpret the results:  

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In two of the reactions you added WT-specific primers to your PCR reaction. These primers
can only bind to the normal version of the gene. If the animal has the normal gene the DNA
of the PCR-reaction will be amplified and you will see a band. If it doesn’t you will see no
band for this reaction. The KO-specific primers, on the other hand, can only bind to the
abnormal, altered transgene.

This means that if the animal has two copies of the normal gene (homozygous normal wild
type animal) you will see a band in the WT-specific reaction, but no band in the KO-specific
reaction. If the animal has two copies of the abnormal gene (homozygous knock out animals)
you will see a band in the KO-specific reaction, but no band in the WT-specific reaction. If
you see a band in BOTH reactions this means that the animals is heterozygous (has one
copy of the normal gene and one copy of the abnormal, altered gene).  

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Appendix:  description  of  materials  used    
LB  (Luria-­‐Bertani  media)  
In 950 ml MilliQ water, dissolve:
• 10 g bacto-tryptone
• 5 g bacto-yeast extract
• 10 g NaCl
Adjust pH to 7.5 with NaOH, adjust total volume to 1l.
Sterilize by autoclaving for 20 min.
 
LB-­‐agar  
• Same recipe as to LB
• Add 15 g agar
• Melt agar into solution in a microwave
Adjust pH to 7.5 with NaOH, adjust total volume to 1l.
Sterilize by autoclaving for 20 min.
 
TENS  solution  (500  ml)  
• 5 ml 1 M Tris-HCl (pH8.0)
• 12.5 ml 20 % SDS
• 5 ml 10 M NaOH
• 0.5 ml 0.5 M EDTA
Adjust volume to 500 ml with MilliQ water
 
PCR  reactions  mixture  (x  1)  
The PCR-reaction contains:

premix (contents se below)+ gene-specific primers (forward and reverse) + DNA sample
Premix 17 µl
contains:
MgCl2 (1.3 µl)
dNTP (0.5 µl)
”hot start” buffer (2 µl)
”hot start” polymerase enzyme (0.18 µl)
H2O (13.1 µl)
WT primers KO primers
P4_WT_F (10 µM) 1 µl P4_KO_F (10µM) 1 µl
P4_WT_R (10 µM) 1 µl P4_KO_R (10µM) 1 µl

DNA 1 µl

TOTAL VOLUME PER


REACTION= 20 µl

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Kontroller

WT K1

KO K2

HET K3

dH2O K4

Primers

P4_WT_F

5-GGAAAGACATGGCTGACTCTG-3'

P4_WT_R

5- CACGCGGTTGTATTTGTAGC-3'

P4_KO_F

5- GGACGCTTGACCAGACAGA-3'

P4_KO_R

5-AGACGGCAATATGGTGGAAA-3'

 
   

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ANVISNINGAR  FÖR  LABRAPPORTER  
 
LABRAPPORT  1:  ”Recombinant  DNA  technology”  

En labrapport per grupp


Labrapporten ska skrivas på svenska
Max två A4-sidor
(Typsnitt: Times 12 punkter, radavstånd 1.5)

Rapporten ska innehålla svar på frågorna:

SYFTE
• Varför görs laborationen/ vad vill man ha reda på?

Utrustning/metod
• Vad innehåller agarplattan och varför?
• Varför använde ampicillin?
• Varför plockade just en singelkoloni?
• Vilken metod används för att isolera plasmiderna?
• Vilken restrektionsenzym använde ni och varför just detta enzym valdes?
• Varför använde ni gelelektrofores metoden och hur fugerar den? Vad har elekitriskspäning,
jonrikt buffer, gelred, loadingdye och stege med kända baspar för betydelse?

Resultat
• Vilken plasmid användes (1, 2 eller 3) och vad är förvätade storlek?
• Har ni fått rätt klyvning?
• Vilken är genen i denna plasmid och från vilken art kommer den?
• Kort info om denna gens funktion.
• Om resultaten inte blev de förväntade, vad kan det beror på? (Felkällor)

   

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LABRAPPORT  2:  “Genotyping  by  PCR”  

En labrapport per grupp


Labrapporten ska skrivas på svenska
Max en A4-sida
(Typsnitt: Times 12 punkter, radavstånd 1.5)

Rapporten ska innehålla svar på frågorna:

SYFTE
Varför görs laborationen/ vad vill man ha reda på?

Utrustning/metod
• Varför använde ni PCR metoden och hur fungerar den? Hur påverkar de
3 olika temperatur i PCR metoden DNA:t, primrer, polymeras och nukleotider?
• Varför anväde ni gelelektrofores metoden och hur fugerar den? Vilken betydelse har
elekitriskspäning, jonrikt buffer, gelred, loadingdye och stege med kända baspar för denna metod?

Resultat
• Vilka resultat fick ni?
• Vad innebär Homozygot WT, Homozygot KO och Hetrozygot?
• Vilka genotyper hade djuren våra prover kom ifrån? (inkl förklaring till hur man kommit
fram till detta).
• Om resultaten inte blev de förväntade, vad kan det bero på? (Felkällor).

INLÄMNING av labrapport 1 och 2


Ladda upp era färdiga labrapporter som ett enda dokument (innehållande både rapport 1 och 2)
på Studentportalen till Filarean kallad “MOLEKYLÄRBIOLOGILABORATIONSRAPPORT”.
Ange gruppnummer i filnamnet samt vilken version av rapporten det datum (ex Grupp1A.v1.2018.2.6).
För inlämningstider, se Studentportalen.
Datorlaborationer i Bioinformatik: mer info fås på plats i datorsalen, se schemat för
datalaboration gällande bioinformatik (preliminär schema 5/2/2018).  

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