cloning impossible - (May/16/2007 )

Hi guys,

I have been trying to clone 10 Kb insert into pcDNA3.1 for months without any succes!!!!
The insert was amplified by PCR (forward and reverse primers were designed to incorporate EcoRI and BamH1 endonuclease restriction sites, respectively),purified, digested and purified again by WIZARD SV Gel and PCR clean up(Promega)

Ligation is done by T4-ligase (USB) O/W at 4°C. The insert :vector ratio is about 3:1 and like negative control I used digested vector without insert.

Afterwords I trasformed XL10 GOLD E.coli from Stratagene; I got colonies (not in negative control), but none of theme were positive clone: contain no insert and some appear different size also!!!!!

Thanks for your help

Lauretta

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To improve your ligation, which seems to be the bad point in all the process, you can try to add PEG in your mixture (it gets the volume busy and packs the vector and insert DNA close to one another)

Heat shock aren't that great for transformation, so I'd advice to switch for electroporation to improve this step as well and insure that any possible positive clone that you get, will actually be transformed in a bacterium.

keep going with the ligation at 4°C since it decreases the molecular shaking and thus improves sticking of DNA ends, you can try a quickligation enzyme which works even better in this 4°C condition.

good luck

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Hi. Are both EcoRI and BamH1 absent in your cDNA sequence?

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Yes, there are!

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You'll have to redesign the primer set then... with REs absent in the CDS. Good luck

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Oh my God!I wrote "there are" instead of "they are"!!!
Aniway both EcoRI and BamHI are absent in cDNA!!!!

I'm sorry and thank you for your interest!!!

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If the RE sites are too close to the end, RE could not cut. You should clone your PCR product first (in pTOPO or pGEMT or pJET1….). Then, digest with both enzymes.

If you are having colonies, may be vector is religated. I think one of the enzymes is not cutting well.

Good luck!!!

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is your vector only ligation control clean?

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I would suggest using a higher ratio of vector: insert and also use higher amounts of DNA for ligation. This can help.

Good Luck !!!

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Ligation-based cloning approaches become incredibly inefficient as the insert and vector sizes increase.

If you intend to continue cloning inserts and vectors of this size consistently, I would look into recombineering based approaches. It's worked well for me, thus far.

Take a look at Datsenko and Wanner or stuff from DL Court's lab. HIs review is here:

http://www.ncbi.nlm.nih.gov/entrez/query.f...l=pubmed_docsum

-Matt

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