Claudin 3 Annealing Temperature - (Jan/24/2015 )

I'm really sorry if this is wrong place but can't find information anywhere!

Primer sequence

FP: GTCCGTCCGTCCGTCCG

RP: GCCCAGCACGGCCAGC

I did bench top PCR at 54 degrees, not so good then reduced to 52 degrees for  qRT-PCR and have to adjust again to get it correct and go even lower. I've been looking at papers and one website suggested 60 which seems way too high...

This is primer3 output for your primers

WARNING: Left primer is unacceptable: High similarity to mispriming or mishyb library/High 3' stability; Right primer is unacceptable: Unacceptable GC content/High similarity to mispriming or mishyb library/High 3' stability

OLIGO            start  len      tm     gc%   any    3'   rep seq 
LEFT PRIMER        109   17   66.30   76.47  2.00  2.00 17.00 GTCCGTCCGTCCGTCCG
RIGHT PRIMER       269   16   67.84   81.25  4.00  2.00 13.00 GCCCAGCACGGCCAGC
SEQUENCE SIZE: 1318
INCLUDED REGION SIZE: 1318

PRODUCT SIZE: 161, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 2.00

Apart from the warnings there it's obvious, that your primers have very high Tm, in Tm such high I would probably use annealing temperatures 60 and even higher.

BUT..

They are both very short and high GC, the suitable GC content is generaly between 20-80 % some even design strictly between 40-60 %. Suggesting that the template itself is very high GC.
Indeed it has, the whole gene has very high GC content, 69%.

So primer design for this gene, has to be adapted for the high GC PCR, including PCR additives like DMSO or betaine to even work. Look up the literature specifically about PCR on high GC templates to optimize.


But that is not the worst thing.

Since your primers are short to even have melting temperature a bit above 60.. the forward primer is non-specific, if you BLAST it you will see it alignts to many different human template RNA, which is bad. Even worse, it alings on several places within Claudin 3 itself, because look at it.. it's basically a repeat!

If you ever make this work, it will be nonspecific. So I would call this a bad primers that are waste of time to optimize.

This is a very very tricky gene to design primers on, so if you are a beginner, it will be a hard work, I would look up if there are not some primers that are in use already by someone else, or just used all the tools in here to design primers that will be specific, at least not in the repeat area. Of course it depends on what you need it for, and if that will be possible to design.