Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
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
Vector types
Vector
Plasmid
Cosmid
10-40 kb
130-150 kb
About 300 kb
200 kb to 2 Mb
Origin of replication
Antibiotic resistance gene (Amp, Kan, Tet, Chl)
(Multiple cloning site)
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 )
Intended use
Purpose
Example
pGEX4T
Eukaryotic promoter
Tag for protein purification or
detection
Eukaryotic selection marker
pcDNA3.1
Reporter gene
pGL3basic
General cloning
pBluescript KS
Restriction enzymes
(endonucleases)
Restriction enzymes
(endonucleases)
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)
1.
2.
3 solutions strategy
1.
2.
3.
1.
2.
3.
1.
2.
3.
Advantages
Cheap
Fast
Disadvantages
Advantages
Disadvantages
Expensive
Gradient centrifugation
1.
2.
3.
4.
5.
Advantages
Disadvantages
Slow
Expensive equipment is needed
High concentrations of EtBr
Concentration measurement
Photometric measurement of DNA concentration
UV 260 nm
Conc=50xOD260
Questions?