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Ligation problems


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#1 anonymous

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Posted 03 November 2001 - 10:00 PM

Hi
I have a 498 bp gene that I PCR'ed out of a small Staph plasmid (pC223). I encorporated unique restriction site tags in the primers (NdeI in the + primer; BamHI in the - primer). I am now trying to ligate it into a NdeI-BamHI restricted pET vector with the correct concentration of (viable) T4 ligase and leaving it over-night at 14C. I am confident my pET vector is viable and well cut; and I have used a QIAquick PCR cleanup on my PCR'ed gene product (after clipping the ends with NdeI-BamHI to make them sticky). [I gave up gel purifying it due to very low yields from TAE gel extraction]. Trouble is I cannot seem to get a ligation! I get transformation efficiencies of 10E5 with a control, but nada with my ligation. Any suggestions? Am I missing something slightly less than obvious.

I'd be glad of any insight..

Go raibh maith agat!

Aoifa

#2 anonymous

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Posted 03 November 2001 - 10:00 PM

I forgot a few details: <p>I have been using a 10:1 molar ratio of PCR product to pET vector (with 250ng of pET vector being used). (0.7:0.7 pmol)

I also tried a 1:1 molar ratio with no luck either.

Go figure?

#3 anonymous

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Posted 04 November 2001 - 10:00 PM

Aoifa:(I had a friend named Aoife from Ireland)

Try the following

1. Increase spermidine concentration in ligase buffer to 1.5 mM2. Increase PEG concentration by 50% of what is already there3. Start the ligation reaction at 37C for 1 h, add more enzyme, move to room temperature for 2 h, add more enzyme, move to O/N 10 degrees C.

Happy colony picking !!

Angel 43[url="http://www.dstf.bigstep.com"][url]http://www.dstf.bigstep.com[/url][/url]


#4 anonymous

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Posted 04 November 2001 - 10:00 PM

Try running your ligation on an agarose to check for high molecular weight products, in other words, to check whether your ligation is the problem or the tranformation.Also, sometimes, if the restriction sites are too close to the ends of your products the restriction endonucleases wont cut. Are you sure that your product has sticky ends.And why are you using so much pET. 50-100ng should more than enough. It's the insert you should be using a lot of.

#5 anonymous

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Posted 27 November 2001 - 10:00 PM

Aoifa,

The problem may be that your primers did not cut with Nde and Bam. Try TA cloning your pcr product and then cutting with Nde and Bam or design your primers with extensions beyond the restriction sites. Often resriction enzymes fail to cut well if the site is too close to the end.

Hiram





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