Problem with Ligation and Transfection

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apees7k
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Problem with Ligation and Transfection

Post by apees7k » Thu Oct 12, 2006 2:51 pm

I am using pET 28b vector to ligate my gene (@1.5 kb). To do so I have designed primers which has EcoRI and XhoI restrictin sites on 3' and 5' ends respectively. I have digested both the vector as well as the primers with these restriction enzymes (Fastdigest, Fermentas) in a single reaction and gel purified the products respectively. I ran a gel from which it is very hard to tell weather digestion was suggessful but it is clear from the vector band that it is linear but could not tell about the PCR product (insert).
First, Can anyone let me know how can I check quickly weather my vector and the insert is digested completely.
I tried to ligate my PCR product into vector using Fermentas fastligat kit (5min) but it failed alomost every times (no single colonies) but in other hand the undigested pET considered as a +ve control agve good transfection colonies with BL21(gold).
Now I am confused how to proceed as I know soemthing is wrong with restriction digestion or ligation.
Please suggest the best possible way to look at this problem and what initiative I should take at this moment.

Thanks.
AP

oppox
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Post by oppox » Thu Oct 12, 2006 7:18 pm

I have had problem with pET28 too, restriction digestion. You can check your cut material on an agarose gel for more bands then one. I left the restriction digestion and used a pET-blue vector, I got the insert in the right place but I couldnt express the gene.
So my second coice was t try a restriction free cloning (search on a article database, there is one from 2004) and there I got both insert and protein.

When I did the RF-cloning I used a pET28 vector with one sticky feet complementary to the trombin site and the other on the T7 terminator site, I tried to be in the range of the article with deletions. But it didnt work so I fixed it my way instead.

good luck

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