problem in ligating 8 kb fragment

bluefrog at ihug.co.nz bluefrog at ihug.co.nz
Tue Apr 30 04:56:34 EST 2002


On 17 Apr 2002 02:35:44 -0700, tkhare at yahoo.com (tarang) wrote:

Is it possible that you can't clone it because it's too large?
Bluescript is a high-copy vector and having 8-10 kb inserts may have
toxic effects on your cloning host.
One of our PhD students has tried almost a dozen times to clone a
cell-surface gene (even a truncated ORF PCR product of 1.5 kb) into
Bluescript and all she's got is nada.

>hi,
>I am trying to ligate 8kb fragment in bluescript vector SK+. I have
>first tried to double digest 10KB fragment with HindIII and SDAI,
>while I digested the vector with pstI and HindIII. I ligated it at 4°C
>with T4 ligase, overnight. Next day I did heat shock transformation in
>JM109 cells. I got colonies. But when I checked for insert it was not
>there.
>Then I tried to put PCR amplified 10KB in pGEMT as well as in TA
>cloning vector pCR2,1. There I can see blue and white colonies but
>again when I checked for presence of my insert, it was not there.
>My agarose gel with PCR product shows one clear and strong band. I am
>using 25ng/µl of ampicillin for SK+ and pGEMT easy vector while I also
>used 25ng/µl of Kanamycin as selectable marker in TA cloning vector
>pCR2,1. In every case I am getting colonies.
>I am checking with colonie PCR, Restriction digestion and PCR on
>plasmid extraction.
>Is there any trick in isolating Plasmid of such a size. I am using
>qiagen plamid Midi extraction kit.
>Waiting to hear expert advice.
>Sincerely
>Tarang




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