primer desighning with RE sites - (Feb/21/2008 )
Hi,
I designed some primers for my work,based on some suggestions i added EcoR1 sequence to those pimes. its sequence is like this,
primer sequence ggtcatgcaatc
enzyme sequence is gaattc
final sequence of primer with RE is
AAA AAA GAA TTA GGT CAT GCA ATC
IS IT CORRECT OR NOT PLEASE HAVE A LOOK ON THIS AND SEND YOUR REPLY TO ME.
THANKS IN ADVANCE.
SVRREDDY,
Ph.D STUDENT,
ITALY.
It doesn't look right! You have a mistake in the RE recognition sequence. Your primer should be GAA TTC GGT CAT GCA ATC. I see that you've also added extra nucleotides in the 5' part of the primer so that the enzyme can cut properly. I'm not saying that it won't work with what you've added, but I normally add something like CTTGA.
According to the NEB catalog under enzyme properties, it looks like EcoR1 would like to have a G or C flanking the digestion site. I'd design something along the lines of ccgGAATTCggtcatgcaatc.
Hi,
Rkay i am writing my primer with RE sequence check is it correct or not
AAA AAA CCG GAA TTC GGT CAT GCA ATC
PLEASE HAVE A LOOK ON THIS AND SUGGEST ME .
THANKS IN ADVANCE.
Usually I am adding 6 A's before the restriction site and so far it worked fine. But I did not able to locate such a detailed information in NEB catalogue. I got NEB's 2007-08 catalog. Could you please refer with the page no. or the web link? Thank you very much......
Rkay i am writing my primer with RE sequence check is it correct or not
AAA AAA CCG GAA TTC GGT CAT GCA ATC
PLEASE HAVE A LOOK ON THIS AND SUGGEST ME .
THANKS IN ADVANCE.
Well two things...
firstly... the template binding sequence ( GGT CAT GCA ATC ) is very very short. When the PCR reaction initially stars, it is this binding sequence that actually makes contact with the template. As a simple rule, the tm of the primer is calculated based only on the sequence that initially binds to the template. 12bp is not enough. The melting temperature is frightfully low. And with so few bps, non specific priming will be a big problem. Increase the number of bp you have for the binding sequence. Try a tm of 58 to 60 Celsius. (23 -30bp depending on sequence)
Secondly the guard sequence ( AAA AAA CCG ) excessively long. You don't need 9bp. 6bp is more then enough. And while it would work, I don't quite agree with using a poly A sequence as a guard. I would rather use a mixed sequence, leaning slightly toward GC richness.