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Ligation: insert & vector are about the same size

t4 ligation heat-shock ligation insert size troubleshooting

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3 replies to this topic

#1 guayapa

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Posted 03 November 2015 - 10:14 AM

Hello everybody,

 

I ligated an insert (3kb) with two different vectors that are both about 3kb. For one of the vectors the ligation worked out, for the other I got only empty vectors. Might this be due to the fact that vector and insert are about the same size? Do you have any other ideas? I tried general troubleshooting as proposed in manufaturer's instrutions.

Thanks in advance,

guayapa



#2 phage434

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Posted 03 November 2015 - 10:22 AM

That's unlikely to be a problem. Can't tell much more without knowing many more details about the vectors, inserts, protocol.



#3 labtastic

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Posted 04 November 2015 - 06:11 AM

If you were to sequence your empty vector, I would suspect it is just your original, intact vector back again...which can only happen if your vector was incompletely digested.

 

Incomplete digestion can occur if you tried to digest too much vector initially, didn't let the digest go for long enough, used a buffer that one of your restriction enzymes wasn't 100% active in, used old restriction enzyme, etc.

 

In general though, ligating a 3kb insert into a 3kb vector should be no problem. I find letting the ligation reaction go overnight can help with the more tricky ligations. At the minimum I let it sit for 2 hrs. The recommended 5min ligation time is rarely suitable for any ligations I've ever done.


Edited by labtastic, 04 November 2015 - 06:14 AM.


#4 phage434

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Posted 04 November 2015 - 01:14 PM

You can eliminate a lot of background by not trying to cut circular DNA (which is difficult to do to completion) but instead use PCR to amplify linear DNA fragments from a circular template. The template can be at very low concentration, and can be cut with DpnI, which cuts at GATC at methylated sites. Since PCR produces unmethylated DNA, you can cut with DpnI to eliminate circular template, and leave the linear DNA alone. This works best with non-compatible restriction sites at each end of the linear fragment, which will fail to ligate without an insert. This is the technique used in Biobrick cloning as described here:

Assembly of BioBrick standard biological parts using three antibiotic assembly.

Shetty R, Lizarazo M, Rettberg R, Knight TF.

Methods Enzymol. 2011;498:311-26.







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