No PCR amplified with long primers - (Jun/12/2012 )
Ambinlab on Sat Jun 30 06:23:35 2012 said:
Thanks.The following primers used:
Fwd: ATAT GCGGCCGC (NOTI) GCCGCCACC (Kozak) ATGXXXXXXXXXXXXXX (Tm annealing part: 52)
Rev: tgaactcgag (XHOI) TTAAGCGTAATCTGGAACATCGTATGGGTA(HA tag) XXXXXXXXXXXXXX (Tm annealing part: 52)
I amplified using PCR. Ran on a gel, looked like I have the right insert. The template used was plasmid containing my gene of interest. I digested and purified the insert and did the ligation. However, my sequencing results showed that I have my insert there but with no tag/kozak. (i.e before ATG i had my RE site)
Just curious, how long is your expected product size / how long the fragment you cloned in to your vector?