Practical molecular biology

8.10-.12.2012
PD Dr. Alexei Gratchev
Prof Dr. Julia Kzhyshkowska
Prof. Dr. Wolfgang Kaminski

Assistants
Tina Fuchs
Martin Hahn
Amanda Mickley
Illya Ovsiy

Course structure
8.10
Plasmids, restriction enzymes,
analytics
9.10
Genomic DNA, RNA
10.10
PCR, real-time (quantitative) PCR
11.10
Protein analysis IHC
12.10
Flow cytometry (FACS)

Groups
Student

Tutor

Alsara, Mohmmad
Ghosh, Sambuddha
Liu, Xiaolei

Tina Fuchs

Netsch, Philipp
Vasilakis, Thomas
Al Said, Samer
Hong, Jian
Manner, Andreas

Martin Hahn

Shan, Shenliang
Wan, Shan
Gu, Song
Lasierra Losada, Maria
Mohammad, Yousuf

Amanda Mickley

Sachindra
Zhang, Juanjuan
Gudima, Alexandru
Lee, Kuo-Ying
Mock, Andreas
Schlickenrieder, Bastian
Zhong, Weiwei

Illya Ovsiy

Literature
Current protocols in molecular biology
Molecular Cloning: A Laboratory Manual,
Third Edition by Sambrook
www.methods.info

Plasmids, restriction enzymes,

analytics
Plasmid is an extra-chromosomal DNA molecule separate
from the chromosomal DNA which is capable of
replicating independently of the chromosomal DNA.
Vector a carrier (plasmid or other type) used for bringing
target DNA fragment into a host cell.

Vector types
Vector

Target fragment length

Plasmid

0-10 kb (total size up to 15 kb)

Cosmid

10-40 kb

P1 artificial chromosome (PAC)

130-150 kb

Bacterial artificial chromosome (BAC)

About 300 kb

Yeast artificial chromosome (YAC)

200 kb to 2 Mb

Plasmids are essential instruments of

molecular biology

Cloning and sequencing of DNA and cDNA fragments

Generation of genomic and cDNA libraries
Expression of recombinant proteins
Generation of mutant proteins
Analysis of regulatory sequences
Gene targeting

Essential vector elements

Origin of replication
Antibiotic resistance gene (Amp, Kan, Tet, Chl)
(Multiple cloning site)

Map of pOTB7 vector

showing Chloramphenicol
resistance gene (CMR),
replication origin (ORI) and
multiple cloning site (MCS)

Optional plasmids elements

Multiple cloning site

Promoter for cloned sequence
Reporter gene
Tag
Regulatory sequences

B g l II (13 )

Sc a I (5 6 8 9 )

M lu I ( 2 2 9 )

Am p r

Sp e I (250 )
N d e I (4 8 5)

P CM V
T7
H in d I I I ( 9 1 2 )
K p n I (9 22)
B a m H I (9 30 )

p c D N A 3 .1 (+ ) E G F P

E c o R I (16 5 6 )

6 13 1 b p

E c o R V (16 6 8 )
B st X I (16 7 8 )
N o t I (16 8 3 )
X h o I (16 8 9 )
X ba I (16 9 5 )

S V 4 0 p o ly A

A p a I (17 0 5 )

B H G p o lyA
S V 4 0 p ro m
Neo

S m a I (2 7 8 1)
E h e I (29 6 9 )

Important plasmid information

Replication origin defines the host bacteria: ColE1

replication origin is required for E.coli
Replication origin may define the number of plasmid
copies per bacterial cell
Bacteria may lose recombinant plasmid during
cultivation due to the absence of partitioning system
(par). Naturally occurring plasmids contain par that
ensures that every bacterial cell contains the
plasmid.

Selection of the plasmid vector

Copy number
Replication origin

Intended use

Replication origin of pBR322 vector Expression of proteins in bacteria.

restricts number of plasmid copies Very useful for toxic protein or
per cell to 30-40.
when tight control of protein
amount per bacterial cell is needed.

Replication origin of pUC vector is

a mutated version of pBR322
lacking Rop/Rom gene and allows
up to 500 copies of plasmid per
cell.

Amplification of high amounts of

plasmid DNA in bacteria.
Expression of high amounts of
proteins in bacteria.

Selection of the plasmid vector

Purpose of use

Purpose

Special vector feature(s)

Example

Recombinant protein expression

in bacteria

Regulated bacterial promoter

Tag for protein purification

pGEX4T

Recombinant protein expression

in eukaryotic cells

Eukaryotic promoter
Tag for protein purification or
detection
Eukaryotic selection marker

pcDNA3.1

Analysis of eukaroytic promoter

Reporter gene

pGL3basic

General cloning

pBluescript KS

Restriction enzymes
(endonucleases)

Cut specific DNA sequence

Protect bacteria from phage infection by digesting
phage DNA after injection
Cellular DNA is protected by methylation that blocks
restriction enzyme activity
Restriction enzyme (RE) means restricts virus
replication
Endonucleases are enzymes that produce internal cut
called as cleavage in DNA molecule

Restriction enzymes
(endonucleases)

Presence of RE was postulated in 1960 by W.Arber

The first true RE was isolated in 1970 by Smith,
Nathans and Arber. In 1978 they were awarded the
Nobel Prize for Phylsiology and Medicine.
RE remain indispensible from molecular cloning and
sequencing.

Restriction enzymes
(endonucleases)
Type I enzymes cut at a site that differs, and is located at least at at least
1000 bp away, from their recognition site.
Type II enzymes recognize sites of 4-8 nucleotides and cleave DNA at the
same site
Type III enzymes recognize two separate non-palindromic sequences that
are inversely oriented. They cut DNA about 20-30 base pairs after the
recognition site.

Restriction enzymes
(endonucleases)
Type I enzymes cut at a site that differs, and is located at least at at least
1000 bp away, from their recognition site.
Type II enzymes recognize sites of 4-8 nucleotides and cleave DNA at the
same site
Type III enzymes recognize two separate non-palindromic sequences that
are inversely oriented. They cut DNA about 20-30 base pairs after the
recognition site.

Restriction enzymes
(endonucleases)

Creating genomic and cDNA libraries

Cloning DNA molecules
Studying nucleotide sequence
Generating mutated proteins

Plasmids, restriction enzymes,

analytics
Gel electrophoresis is a technique used for the separation
of deoxyribonucleic acid (DNA), ribonucleic acid (RNA),
or protein molecules using an electric current applied to
a gel matrix.

Ethidium bromide stained agarose gel of

total RNA (1-3) and DNA ladder (M)

Plasmid preparation stage 1

1.

Plasmid-containing bacteria are cultivated in liquid

media, supplemented with the antibiotics for 18 h at
37C with intensive shaking

2.

Cells are harvested by centrifugation

Preparation of the lysate

3 solutions strategy
1.
2.
3.

Resuspend in hypotonic buffer with RNase (buffer P1)

Lyse bacteria using NaOH/SDS solution (buffer P2)
Neutralize NaOH and precipitate proteins using NaAc
buffer (buffer P3)

Plasmid can be isolated from obtained lysate using

various strategies.

Possible methods for isolation

1.
2.
3.

Ethanol or Isopropanol precipitation

Silica matrix bind-wash-elute procedure
Density gradient centrifugation

Precipitation quick and dirty

Also known as mini prep

1.
2.
3.

Ethanol is added to the lysate

Obtained sample incubated for 30 min
DNA is collected by centrifugation

Advantages

Cheap
Fast

Disadvantages

Small amounts of DNA

Poor purity, not sufficient for
applications like transfection
and in vitro translation
Concentration of the
plasmid can not be
determined photometrically

Silica matrix columns

1.
2.
3.
4.

Apply lysate on the column

Wash the column
Elute the plasmid
Precipitate

Advantages

High purity of the plasmid

Fast

Disadvantages

Expensive

Gradient centrifugation
1.
2.
3.
4.
5.

Mix lysate with CsCl solution

Add EtBr
Centrifuge in the ultracentrifuge for 12-36h
Collect the plasmid
Precipitate

Advantages

The very best plasmid

purity
Relatively cheap

Disadvantages

Slow
Expensive equipment is needed
High concentrations of EtBr

Concentration measurement
Photometric measurement of DNA concentration
UV 260 nm
Conc=50xOD260

Important! Photometric measurement of DNA concentration can not be

applied for quick and dirty plasmids, because of the presence of
RNA rests.

Gel electrophoresis of plasmid DNA

Selection of agarose concentration

Plasmid on an agarose gel

Questions?