Transcript Practical molecular biology

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
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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)
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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
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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
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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
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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
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Multiple cloning site
Promoter for cloned sequence
Reporter gene
Tag
Regulatory sequences
Bgl II (13)
Sca I (5689)
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Mlu I (229)
Spe I (250)
Amp r
Nde I (485)
P CMV
T7
Hin dIII (912)
Kpn I (922)
Bam HI (930)
pc DNA3.1(+) EGFP
Eco RI (1656)
6131 bp
Eco RV (1668)
Bst XI (1678)
Not I (1683)
Xho I (1689)
Xba I (1695)
Apa I (1705)
SV40 poly A
BHG polyA
SV40 prom
Neo
Sma I (2781)
Ehe I (2969)
Important plasmid information
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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
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pBluescript KS
Restriction enzymes
(endonucleases)
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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)
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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)
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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
37°C with intensive shaking
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Cells are harvested by centrifugation
Preparation of the lysate
3 solutions strategy
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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
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Ethanol or Isopropanol precipitation
Silica matrix bind-wash-elute procedure
Density gradient centrifugation
Precipitation “quick and dirty”
Also known as mini prep
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Ethanol is added to the lysate
Obtained sample incubated for 30 min
DNA is collected by centrifugation
Advantages
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Cheap
Fast
Disadvantages
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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
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Apply lysate on the column
Wash the column
Elute the plasmid
Precipitate
Advantages
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High purity of the plasmid
Fast
Disadvantages
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Expensive
Gradient centrifugation
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Mix lysate with CsCl solution
Add EtBr
Centrifuge in the ultracentrifuge for 12-36h
Collect the plasmid
Precipitate
Advantages
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The very best plasmid
purity
Relatively cheap
Disadvantages
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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?