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pcDNA 4/TO

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

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pcDNA 4/TO

Postby am1795 » Mon Jan 12, 2009 6:58 pm

I have retrieved a plasmid (pucSIVGAG) that contains the insert Sivgag. I digested the plasmid using Sma I and did a gel purification to isolate the insert sivgag.

I am inserting sivgag into plasmids known as pcDNA 4/TO and pcDNA 3.1zeo+. I recently added Not I linkers to the ends of sivgag and purified with Promega PCR purification kit. I isolated the pcDNA vectors from gel purification and digested both insert and plasmid with Not I enzyme. I ligated the vector and inserts and transformed the ligation into EColi competent cells. I plated the cells on Carbenicillin LB Agar plates.

I have done this several times yet on all occasions there are no colonies that grow on the plates. If there happens to be colonies I run minipreps on them and I see that the vector has self-ligated and the insert is not being taken up. Is there something else I can do to get the insert to be taken up? Or is there something I am doing wrong?

Note: I have also already done treatment of shrimp alkaline phosphatase on the vectors to remove the phosphates and lessen the occurrence of self-ligation
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Postby GreenDog » Tue Jan 13, 2009 10:56 am

I don't understand much in this but I think it would have been better if you had ends which are not complementary on the plasmid to prevent it from closing on itself. Maybe blunting one end of the plasmid and the corresponding end on the insert and using a ligase which connects blunt ends.
Also are you sure you are getting nothing after the miniprep maybe the bend in very light but you are still getting something, could you show the gel?
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