Cloning problem!! In desperate need of help!!

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eneleda
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Cloning problem!! In desperate need of help!!

Post by eneleda » Mon Jun 12, 2006 5:36 am

Hi, I am trying to make a hairpin RNA construct. The method I use is to place
2 different cDNA (sense sequence and antisense sequence) into a plasmid and then transcribe it to form the hairpin RNA. The sense sequence has been ligated into the vector using EcoRI and SacI cut sites but the antisense sequence which has SacI and PstI cut size (the SacI site of the antisense sequence should ligate to the SacI cut site of the sense sequence which is already in the vector) refuses to go in. I digest the vector (containing the sense sequence) and the antisense sequence (which is the insert) with Sac1 and Pst1, run in on a gel and gel purify it, ligate it togetehr and transform but I never get any transformants. In fact, my plates are so clean it looks as if I did not plate anythg on it. What is going on here? The ligation seems to be working cuz I have tried running the ligation mixture on a gel to check. The only way I can get cells to grow is if I do not gel purify the vector (which means the fragements are not cleaned after digestion) but of course I get loads of false positives which are impossible to screen. Please help!! I have been trying for months!

weesper
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Post by weesper » Thu Jun 15, 2006 12:05 pm

I'd try and find a new vector.

herb386
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Post by herb386 » Thu Jun 15, 2006 8:17 pm

Follow the last link on this page

http://www.freewebs.com/ads-online/

Then go to ligation reaction and scroll down to find lots of troubleshooting information.

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