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Help for pGEM-T easy vector ligation problem


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#1 biovinz

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Posted 10 March 2013 - 11:36 PM

Hi all,

I have a trouble with ligation of my insert in pGEM-T easy vector. After transformation, I got blue and white colonies. so i took white colonies and did enzyme digestion but there is no insert..i tried ligation for 1hr at RT, overnight at Rt and overnight at 4C..after that no difference, there is only vector..

Please help me...

Thanks in advance

vinod


#2 GNANA

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Posted 11 March 2013 - 04:28 AM

make sure your Polymerase have terminal transferase activity.....otherwise you just gotto optimize the vector and insert ratio....sometimes purifying the PCR product helps....
I would prefer being perfectionist rather than a passionist in Research.

I always had an alternate hypothesis....

#3 biovinz

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Posted 12 March 2013 - 03:46 AM

Thank you so much for your reply....




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