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PCR

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PCR

Postby akane19861020 » Thu Dec 15, 2011 9:39 am

I need help.

I design primer for PCR:

Forward primer: at cag tCC ATG gga aca ggc tca caa g [CCATGG is NcoI restriction site]
Reverse primer: tat GTCGACTTA TTC GTG CCA TTC GAT TTT CTG AGC CTC GAA GAT GTC GTT CAG ACC GCC ACC CCA AAT GGC TCC C [GTCGAC is SalI restriction site]

Because I need to add an Avitag to C-terminal of my protein, so I designed in that way with 2 RE cutting site.

However, after PCR, double digestion, ligation and transformation, I got lots colonies, however, none is the positve result.

Could somebody help me with this matter?

Thanks!
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Postby JackBean » Thu Dec 15, 2011 12:25 pm

did you get nice sharp band after the PCR? If so, I wouldn't suspect PCR being faulty. However, your reverse primer is quite long.
How did you do the double digest, ligation and transformation?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Re: PCR

Postby genetherapy » Thu Dec 15, 2011 5:05 pm

Agree with Jackbean,
Reverse primer is too long. Primers are usually 20-30 nucleotides long. Your reverse is about 76 nucleotides. Also it is better to check your PCR, digestion etc. with agarose gel so you can go on controlledly and find which step the problem is.
Good luck
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Re:

Postby akane19861020 » Fri Dec 16, 2011 1:12 am

Yes, I finally checked out it is the problem of primer. Thanks a lot!

JackBean wrote:did you get nice sharp band after the PCR? If so, I wouldn't suspect PCR being faulty. However, your reverse primer is quite long.
How did you do the double digest, ligation and transformation?
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