Biology Forum › Molecular Biology › PCR
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- December 15, 2011 at 9:39 am #15846
akane19861020ParticipantI 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!
- December 15, 2011 at 12:25 pm #108736JackBeanParticipant
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? - December 15, 2011 at 5:05 pm #108753genetherapyParticipant
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 - December 16, 2011 at 1:12 am #108758akane19861020Participant
Yes, I finally checked out it is the problem of primer. Thanks a lot!
quote JackBean: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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