Cloning DNA with E.coli
Only one
DNA in each
bacterium
One bacterium
Gives one
colony
1
2
SELECT
3
4
5
Competent Cells
Recovery
In broth
6
Optimize ligation
All molecules depicted
are ds DNA
May be worth
purifying desired
DNA fragment
first on a gel
Find the right product
7
(if ori and antibiotic resistance present)
8
Prevent vector self-ligation - Alkaline Phosphatase
9
Convert to blunt ends - DNA polymerase (s)
Klenow
T4 DNA pol
10
Homopolymer tailing
11
12
Adaptors - changing the overhang
13
Linkers blunt to sticky
ends of choice
14
Lambda Phage
15
Lambda phage genome
16
17
Packaging the lambda genome
18
19
Packaging excises recombinant phage from complex DNAs
20
Plaques
+ soft agar
Pour on hard agar & incubate
21
20 kb
5 kb
2 kb
0.5 kb
“6-cutter”
“4-cutter”
22
Genomic fragments
For a library
23
3’
C
GAGCT
5’
5’
TCGAG
C
3’
CTC
GAGCT
TCGAG
CTC
GATCN
N
GATCN
AGN
N
NCTAG
NGA
NCTAG
CTC GATCN
GAGCTAGN
24
Vector XhoI
Fill with dC & dT
Insert Sau3A
Fill with dG & dA
Ligate
3’
C
GAGCT
5’
5’
TCGAG
C
3’
CTC
GAGCT
TCGAG
CTC
GATCN
N
N
NCTAG
GATCN
AGN
NGA
NCTAG
CTC GATCN
GAGCTAGN
25
cDNA libraries
26
Insertion
vector
in HFL strain host
efficiently in HFL strain host
27
Size of a representative genomic library
28
BAC vectors
+
Inducible
Activator
of oriV
29
30
YACs
31
PCR
32
T
B
T
B
T
B
T
B
T
B
33
34
35
PCR Enzymes; speed, accuracy & ends
36
Primer 1
RT-PCR
Primer 2
37
Primer 1 & 2 or nested primers
Cloning PCR products
TA cloning
38
39
40
41
42
Lambda recombination system used for cloning
100bp
168bp
43
+
P
P1----P2
+
+
B
B1----B2
BP
+
LR
L
L1----L2
+
+
R
R1----R2
44
Recombination cloning of PCR product to ENTRY vector
+ R1-ccdB-R2
45
ENTRY vector to DESTINATION vector(s)
46
MAGIC
47
48
Ligation independent cloning (LIC)
Add complementary sequences at ends of each fragment by PCR
Chew back with T4 DNA pol, adding one nucleotide
to dictate stop site and expose complementary strands
Anneal and transform E. coli
49
SLIC
Unequal ssDNA lengths increase versatility
(no specific sequence, complementarity can be longer to increase specificity & efficiency)
50
More SLIC
Multiple fragments can be joined together reasonably efficiently
provided they all have different complementary regions at each end
51
In vitro
Site-specific
mutagenesis
Dut = deoxyuracil
phophotransferase. Dut- cells
accumulate dUTP--> DNA
Ung = uracil N-glycosylase.
Ung- mutants do not excise uracil
from DNA
Growth of plasmid template
plasmid on dut- ung- E. coli double
mutant cells gives U in DNA
Synthesize mutant DNA using
Transfect heteroduplex into
dut+ung+ E. coli.
U gets removed from WT
template strand, leaving
apyrimidinic sites, these cannot
replicate.
Mutant synthetic strand survives
normally.
“Quik-change”
Has mA at GATC
Linear
Amplification
Uses only
Original DNA
Dpn1 cuts only if mA
present in at least
one strand of GATC
Newly synthesized
DNA does not have
mA at GATC