Question about PCR primer with degenerate base at 3' end - (Dec/19/2005 )
hi, I just have a question,
I recently order a fluorescent labeled primer to ABI (applied biosystem)
and sequence was like
5'- -------RY-3' (R is purine, Y is pyrimidine)
and next day, they told me they cannot make promer ended with Y.
so, I asked why? to tech suppot, but what they told me is they can make
primer ended either T or C, but cannot combine 50:50 mixture
so, does anyone have any idea why they cannot make the mixture?
beside, I ordered the exactly same primer to qiagen (operon),
and they had no probrem to making it.
but, if 50:50 mixture itself have any probrem, I probably shouldnt use the primer
that I got from operon, right?
any suggestion or idea help me, so if you have anything in your mind, please help me to understand!!!
Many synthesizers use a 3' base which is already attached to glass beads on the column. These come in each of the four base types, but not in mixtures. So, mixed bases are possible in any position except the 3' position. Of course, you could have made two primers, one with T and one with C at the 3' end, and mixed equal amounts of them. Qiagen must use so-called "universal" columns, which have no 3' base already incorporated.
thank you for the reply phage434, so do you think the difference between two company would be just how they made primer, right
I was just wondering that the mixing of primers might be worng in this case or something
I will use the primer from operon and see what's gonna happen.
Thank you again