Revision as of 19:46, 16 September 2011

Ligation

After following our digestion protocol a ligation can be performed.
Different ratios of vector:insert are used, to get the best result out of the ligation.
To check for self-closing vectors a vector-only sample will be ligated.

Materials

PCR tubes/PCR plate
vector and insert DNA
dH20
T4 DNA ligase
T4 DNA ligase buffer

Note: All materials should be held on ice during preparation.

Protocol

ratio 1:1	ul
Vector	1
Insert	1
T4 DNA ligase buffer	1
Ligase	1
H2O	6
Total	10 ul

ratio 1:2	ul
Vector	1
Insert	2
T4 DNA ligase buffer	1
Ligase	1
H2O	5
Total	10 ul

ratio 1:5	ul
Vector	1
Insert	5
T4 DNA ligase buffer	1
Ligase	1
H2O	2
Total	10 ul

Vector-only	ul
Vector	1
Insert	0
T4 DNA ligase buffer	1
Ligase	1
H2O	7
Total	10 ul

1. Incubate for ON at 16C.
2. Incubate for 20min at 80C to heat kill.
3. Use 2ul of ligation to transform into competent cells.

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 . Use 2ul of ligation to transform into competent cells.<br>
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