8 replies to this topic

[lucabag]

I must subclone a gene from a vector to another.
I designed PCR forward and reverse primers containing BamHI and PSTI restriction sites, respectively.
PCR resulted in an expected 1750 bp product.
After "gene clean" I digested with the two restriction enzymes both as single cut (just PSTI or BamHI) and double cut, expecting a product shorter of about 10 to 25 bp.
Result: digested products run slower than undigested in all the cases....They seem longer and heavier than undigested product!
Double digested seems longer than single ones!!!!
I tried several time changing enzyme brand, digestion duration, and agarose gel percentage.
I always add BSA (as requested) in the digestion mix.
Someone can explain to me this?
By the way cloning was always unsucessfully :-(((

Thank you

[gt_ameya]

Lucabag,

Even I have seen this phenomenon where the digested product runs slower than the undigested one. I think, somehow, the buffer we put in makes it run slower. But thats just a guess and somebody will correct me, if I am wrong.

Do you clean up your digests before the ligation?

[lucabag]

gt_ameya, on 07 March 2011 - 01:43 AM, said:

Lucabag,

Even I have seen this phenomenon where the digested product runs slower than the undigested one. I think, somehow, the buffer we put in makes it run slower. But thats just a guess and somebody will correct me, if I am wrong.

Do you clean up your digests before the ligation?

Thank you for the suggestion! In this moment I'm trying to digest without BSA since I had the same feeling!

Yes I always clean the band from the gel with gene-clean column kit.

thank you

Luca

[phage434]

REk's can sometimes stick to the substrate DNA, making slower running fragments. Normally, I'd suggest heat killing the enzymes, but here BamHI cannot be heat killed. As has been pointed out, salt concentrations from different buffer conditions have a large effect. In any case, seeing digestion of 10-15 bp on a 1700 bp fragment will be very challenging. You can test cutting and religation by ligating the insert only and looking for multimers. If you cut only one end (or add individual enzymes to the ligation mix) you can observe the double length fragment and quantify the amount that can be cut and ligated, independently for each enzyme.

[lucabag]

phage434, on 07 March 2011 - 05:05 AM, said:

REk's can sometimes stick to the substrate DNA, making slower running fragments. Normally, I'd suggest heat killing the enzymes, but here BamHI cannot be heat killed. As has been pointed out, salt concentrations from different buffer conditions have a large effect. In any case, seeing digestion of 10-15 bp on a 1700 bp fragment will be very challenging. You can test cutting and religation by ligating the insert only and looking for multimers. If you cut only one end (or add individual enzymes to the ligation mix) you can observe the double length fragment and quantify the amount that can be cut and ligated, independently for each enzyme.

Thank you for suggestion!
I single cut and re-ligate the PCR products: gel response isn't so clear... I obtained some longer (larger) fragments compared to the original PCR product but not the exact double size band, as expected. Cutting just one end at a time I don't see any reason to obtain multimers. What do you think?

[phage434]

There's no reason you should see multimers. You should heat kill the ligase before electrophoresis, and make sure you are not using PEG in your ligation buffer (no "quick ligase" buffer).

[lucabag]

phage434, on 08 March 2011 - 10:21 AM, said:

There's no reason you should see multimers. You should heat kill the ligase before electrophoresis, and make sure you are not using PEG in your ligation buffer (no "quick ligase" buffer).

Thank you I'll try

[truta]

I've read in the NEB catalog somewhere that you can add 0.5% SDS to your sample after the digest to release the enzyme from the substrate.

Edited by truta, 12 May 2011 - 12:57 AM.

[Evanescence]

I guess you need to check the manufacture recommendation, Using NEB is flexible for me,
Are you doing sequential digestion i mean digest the plasmid with the enzyme one by one..
i agree with phage could be salt concentration..

Your cloning is not successful? maybe because of the your ligation buffer is too concentrated or diluted, the digestion is not good or maybe the something wrong with the mixture, Maybe you can place your ligation mixture at lower temperature than u used before, overnight ligation should be produced good result..Good luck and keep trying let us know once u find out the solution