Corum:T4 Ligation: Difference between revisions

Latest revision as of 15:05, 30 August 2012

Standard DNA ligation using NEB T4 ligase to form a vector.

For a 10 μL ligation reaction:

3 μL sterile ddH₂O
1 μL 10X ligation buffer
5 μL ~100nM linker dsDNA (either hybridization product or digested, purified PCR product)
0.5 μL ~10nM backbone dsDNA (gel extracted digestion product from donor plasmid)
0.5 μL T4 ligase

(Note: ratio of backbone to linker given here is ~1:100. This may need to be adjusted in some cases.)

Add the reaction components together in order listed above. Mix gently and spin.
Include -linker control reaction (sub sterile ddH₂O for linker).
Incubate 16 °C overnight (8 hr minimum).
Transform 2.5 μL rea(or more, if necessary, to obtain an acceptable amount of colonies) into chemically competent cells.

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

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*SC 18:03, 30 August 2012 (EDT): Removed 1mg/ml BSA component from protocol, per the fact that it is not recommended in the NEB protocol.

@@ Line 9: / Line 9: @@
 For a 10 μL ligation reaction:
+* 3 μL sterile ddH<sub>2</sub>O
+* 1 μL 10X ligation buffer
 * 5 μL ~100nM linker dsDNA (either hybridization product or digested, purified PCR product)
 * 0.5 μL ~10nM backbone dsDNA (gel extracted digestion product from donor plasmid)
-* 2 μL sterile ddH<sub>2</sub>O
-* 1 μL 10X ligation buffer
-* 1 μL 10mg/ml BSA
 * 0.5 μL T4 ligase
-(Note: standard ratio of backbone to linker given here is ~1:100. This may need to be adjusted for non-standard conditions.)
+(Note: ratio of backbone to linker given here is ~1:100. This may need to be adjusted in some cases.)
 ==Procedure==