Gene cloning, a practical step by step overview

1

• Lecture 5
• Gene cloning, a practical step by step overview
• Learning outcomes
• You should be able to understand
• the practical steps involved in gene cloning
• how to purify and prepare plasmid and insert DNA
  for cloning
• how to prevent vector self ligation and multiple
  insertion
• how to transform a ligation product into E. coli
• how to select for recombinant DNA molecules
• how to analyse a clone using restriction enzyme
  digestion

2
Gene cloning, a practical step by step
overview 1. prepare plasmid (vector) DNA and
DNA to be inserted 2. digest with restriction
enzyme 3. ligation 4. Transformation into E.
Coli 5. Selection and identification of
successful clones
3
Basic steps of cloning
cut ligate
E. coli

Vector
transform

Insert DNA
Replication
extract and purify
Bacterial division
4
Prepare plasmid (vector) DNA Plasmids are
commercially available, or can be obtained from
colleagues Plasmid DNA can be transformed into
E. coli, amplified and isolated from bacterial
DNA/RNA/protein
5
Purification of plasmid DNA separation from
bacterial genomic DNA Example Alkaline Lysis
ssDNA
supercoiled plasmid
Linear DNA
pH 12
pH 7
single strand linear DNA aggregate
Centrifugation
6
Purification of plasmid DNA from bacterial
protein, RNA and other contaminants
DNA RNA
centrifugation
Mix with phenol
interface (denatured protein)
Phenol
contaminating RNA can be degraded using RNase
7
Preparation of insert DNA Dependent on source
of DNA PCR Partial digestion (to create
library) cDNA etc....... More examples in
future lectures......
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• Restriction digest of vector DNA and insert DNA
• mix together
• DNA to be cut
• Restriction buffer 110 of final volume
• water
• restriction enzyme
• incubate 1 hr (or more) at 37 ºC (for most
  enzymes)
• Points to consider
• 1 unit of restriction enzyme cuts 1 µg of DNA per
  hour, more enzyme is needed when DNA is dirty
• Choose buffer which provides optimal conditions
  for specific enzyme

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Wanted and unwanted ligation outcomes
Self ligation of vector
Single insert
Multiple inserts
10
Optional prevent self-ligation of vector through
de-phosphorylation
5'
5'
3'
3'
OH
P
HO
P
5'
5'
3'
3'
Calf Intestine Phosphatase (CIP)
5'
5'
3'
3'
OH
HO
Not ligatable!!!
HO
OH
5'
5'
3'
3'
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De-phosphorylated vector can still ligate with
phosporylated insert
5'
5'
3'
3'
OH
P
HO
OH
5'
5'
3'
3'
5'
3'
HO
OH
5'
5'
3'
3'
Non-ligatable nick will be repaired in E. coli
12
Wanted and unwanted ligation outcomes
Prevented by de-phosphorylation of vector or
non-compatible ends
control by vectorinsert ratio
Self ligation of vector
Single insert
Multiple inserts
13
Insertvector ratio is important for successful
cloning
low ratio high chance of vector self-ligation
high ratio high chance of multiple insertion
11
21
31
41
51
Optimal for dephosphorylated vector
Optimal for cohesive end ligation
Optimal for blunt end ligation
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Ligation Mix together Vector and insert DNA
to test tube, use optimal ratio Ligase buffer 1
10 (contains ATP) Water Ligase Incubate 16 ºC
O/N or 1 hr RT
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Transformation of ligated DNA into E. coli
plasmid DNA
CaCl2 treatment
Competent E. coli
E. coli
Heat shock 42 ºC
Transformed E. coli
1 hr 37 ºC Allow expression of antibiotic marker
16
Select for cells that received plasmid
Lawn of untransformed bacteria
Transformed bacteria form colonies
17
Select colonies that contain a plasmid with
insert DNA Blue/white screening (Lecture 1)
18
Identify colonies with recombinant DNA
molecules Pick (white) colonies and isolate
plasmid DNA (mini-prep) Use restriction digest
to determine presence and size of
insert Reverse of ligation use restriction
enzyme to excise insert DNA Run on gel and
determine size of insert
marker
?
linearised vector
Excised insert
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Using restriction digests to analyse recombinant
clones 1. determine presence and size of insert
E
B
EcoRI (E) BamHI (B) HindIII (H)
clone unknown BamHI fragment
2.7 kb
B
H
cut with BamHI
marker
8
7
6
5
5 kb insert
4
3
2.7 kb linear vector
2
20
Using restriction digests to analyse recombinant
clones 2. map internal Sal1site
marker
E
8
B
7
6
5
4
cut with BamHI SalI
3 kb insert
3
2.7 kb linear vector
B
2
2 kb insert
H
SalI
BamHI
BamHI
2 kb
3 kb
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Using restriction digests to analyse recombinant
clones 3. determine insert orientation
EcoRV
EcoRV
E
E
B
B
1 kb
1 kb
SalI
or
SalI
B
B
H
H
EcoRV SalI double digest
marker
8
7
6
5
4
3
2