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cloning not working for months!!!! need suggestions..

cloning not working for months!!!! need suggestions.. - Molecular Cloning Forum

cloning not working for months!!!! need suggestions.. - DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


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Old 06-23-2011, 11:52 PM
Ria Ria is offline
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Question cloning not working for months!!!! need suggestions..



Hi,

I have been trying to clone a ~900 bp Drosophila gene into pUASTattB vector. It is not so hard, as it seems, but it's just not working.

Here's what I have done:

Insert: PCR product (purified) of the gene with 5' NotI and 3' XbaI sites, incorporated while PCRing (with those sites on the 5' and 3' primers respectively).

Vector: pUASTattB

Double digestion: Insert and vector doubly digested with NotI and XbaI (I know that XbaI is sensitive to dam site overlapping, and I have checked and found that there is no dam site overlapping in the vector.) I have gel purified and checked the products on a gel and found there is sufficient DNA in the purified products. I used0.5- 2 microlitres of each enzyme in a 50 microlitre of total digest volume. I have tried all sorts of incubation periods from 2 to 16 hours at 37C. I have also tried with and without phosphatase treatment of the digested vector.

Ligation: I have set up a ligation of 20-60 microlitre volumes with 3:1 insert:vector ratio and used 0.2-1 microlitre T4 ligase. I have also set up reactions of empty vector with or without ligase for negative controls. Incubations were at 14C overnight.

Electroporation: I have used 2 microlitres of the ligation mixture to electroporate 20 microlitres of DH5alpha electro-competent cells. Sometimes I have done salt-precipitation of ligation product and sometimes without salt-precipitation. But, never did I got colonies on my plates!! My negative control (mock transformed) and positive control (transformed with the uncut pUASTattB vector) worked efficiently. But, no success with my experimental plates.

I have done these steps so many times by altering the protocols, incubation periods etc. I have used enzymes from NEB and a few times used ligase from Fermentas.

Please give me your suggestions.

Thanks,

Ria
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Old 07-25-2011, 07:12 PM
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Default Re: cloning not working for months!!!! need suggestions..

Possibly a silly question for you but when you say you added the restriction ends to your primers did you also include an 4-6 bp tail on the end of that?
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Old 08-08-2011, 10:14 AM
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Default Re: cloning not working for months!!!! need suggestions..

Hi, I normally treat my digested vector with calf intestinal alkaline phosphatase then only purify them. Usually the critical part is the ratio between vector and insert, for my case, i have a successful ligation with 6:1 insert:vector ratio. also, once, i discovered that the problem was with the T4 ligase, after changing to new T4 ligase, everything went perfect.
Good luck then
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