cloning troubles

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jamiejlg
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cloning troubles

Post by jamiejlg » Sun May 13, 2007 1:53 pm

Thanks for your answers from my last post.
I'm having trouble with a cloning that shouldn't be so difficult, but I'm not getting transformants. Do any of you have suggestions for the following?

I'm synthesizing a 230 bp fragment through PCR using overlapping oligos. I got a PCR product that appears to be the right size. Although I want to sequence the PCR product for verification, I was asked to clone it first. I cut the product with BglII and HindIII, purified, treated with T7 endonuclease I and re-purified. I cut my Kan-selective vector with BamH1 and HindIII and purified. OD's look ok. I ligated and transformed with One Shot cells and nothing. bummer.
Does anyone have suggestions?

Thanks,
Jamie

seaseasea
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Post by seaseasea » Sun May 13, 2007 2:17 pm

In my opinion, I think it's may be difficult to digest PCR products with REase when no extra bases existing in the terminal of primers. I donlt know whether no growth on plates were caused by this.
Moreover, I advice you clone the PCR products directly into cloning vectors to make a sequence.
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SororSaudade
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Post by SororSaudade » Sun May 13, 2007 10:57 pm

like seaseasea mentioned, if you don't have at least 2 or 3 extra bases after the restriction sites in your primers, the enzymes may not recognize it, hence don't cut... that could be why you're not getting any transformants.

Have you tried different vector:insert ratios?

Just a question... why do you use the T7 endonuclease I?

blcr11
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Post by blcr11 » Mon May 14, 2007 12:16 am

As has already been said, most likely the problem is with incomplete enzyme digestions. Design your end-most primers (the ones with the RE sites) to have at least a 3-base extension. Some enzymes absolutely require extra bases, others are less restrictive. Check out the New England Biolabs or Promega (for two) websites to see which enzymes are most affected by this.

The T7 endonuclease step is an attempt to get rid of incorrect sequences that may have occurred during PCR. You denature/renature the products to "reveal" any mistakes as mismatches which T7 endo then cuts into smaller pieces that are effectively removed by gel purification leaving only correct sequence material in the band of interest. For such a short fragment, if you used a high fidelity polymerase, I wonder if you could skip the endo step. Your choice, though.

SororSaudade
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Post by SororSaudade » Mon May 14, 2007 12:12 pm

thanks bclr11 for your answer :)

jamiejlg
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Post by jamiejlg » Wed Jul 11, 2007 5:22 pm

just an update for anyone who might have wondered and the nice people who offered suggestions. I was able to subclone my 230 bp gene fragment with Invitrogen's zero-blunt kit and then insert it into my desired vector. I did a side by side sequence verification with T7 endonuclease treated and without. I found the endonuclease treated PCR products to have lots of mutations! Non-endo treated PCR products came out great. Kudos to Novagen KOD polymerase.

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