No band on BSP - (Jul/28/2005 )

As long as the primers have a high Tm (57-60C) and are within 2C of each other, that is all. The length can vary, I typically pick primers that are 30mers in length.

Nick

Somewhat curious, which method do you use to calculate the Tm of your primers? Tm= 64.9 +41*(#G+#C-16.4)/(#A+#T+#G+#C) or the equation by Rychlik, Spencer and Rhoads (Nucleic Acids Research, vol 18, num 21, pp 6409-6412) which is what Primer 3 utilizes.

The reason I ask is that the company which we normally order our DNA oligos from apparently calculates it using the conventional method and the Tms which they list on our ordered primer sets are almost always much lower (3-5C) than the designed Tm from primer 3. Which of the two would you say is more accurate?

There are many ways to skin a cat like there are many ways to calculate Tm of primers.

Primer3 I find is more accurate. However, I routinely perform PCRs with Tm 2C lower than that calculated by Primer3.

Our primer synthesis company gives us a Tm value at a set salt concentration, and the salt conc does apparently affect Tm of the primer.

This could be why you are getting the discrepancy.

Nick

Hi,

I am wondering if anybody could help me to design BSP primers. I couldn't figure out how to use the perlPrimer.

Thanks a lot!

Here is the sequence.

ctgctcta agcttgtaaa ctgtttctgc
ttaagaggga ctgagtcttc agtcattgct ttagggggag aaagagacat ttgtgtgtct
tttgagtaca tgttgtctgg gtcagctcac atttaagctt ttgcttgaaa actagtaaaa
gaaaaatgtt gcctgttaac caataatcat agagctcatg gtattttgag gaaatcttag
aaaacgtgta tacaattgtc tggaattatt tcagttaact gttagttgag gtactgatgc
tgtctctact tcagttatac atgtgggttt gaattttgaa tctattctgg ctcttcttaa
gcagaaaatt tagataaaat ggatacctca gtggtttttt ggtgggttta atatagaagg
aatttaaatt ggaagctaat ttagaatcaa gtaaggaggg acccaggcta agaaggcaat
cctgggattc tggaagaaaa gatgttttta gttcagctca ttccagctca cggtcagctc
agctcagcct aacccagctc acaccagccc agcccagcct attccagcct agctctgctc
atcccagctc attccagctc agcccagcct aacccagctc acaccagccc agcctattcc
agcctagctc tgctcatccc agctcattcc agctcagccc agcctaaccc aggtaacact
agtccagccc agcctattct agccaatccc agctcattcc agctcagctc agcctagccc
agctcacacc aggccaggcc cagcctattc cagcctagtt cagcccatcc cagctcattc
cgctcagctc agcctagccc agctcacacc agcccagccc agcccagcct attccagcct
agctcagctc agcccagccc agctcaggca gcctagcttg atagccagtt attgagccat
ccaaccctag tcagtcaggt cagcacagac catgccaatc tagtttagct agtccagtcc
agtttattcc agctcagcac aacccaccca gcccagccca gcctggccta gtttgggtag
cctagtctaa tttaacccca ttcagctgga gctcagctag tccattctac cctagcccag
accatgccag agtagctcag ctcatcccag ctcattatgg ttcatctcag ccaagcttag
ctcaggcagc ctagcttagc ctagcctatt ccagcctagc tcagtaagtc tagcctagct
catctcagct taatttagct tagcccagct cagtccactc atcccagatt agccacctgc
ggtgactgat atacgcaggg caagaacaca gttcagccga gcgctggcgc ccgaacaacc
gtacagaaag ggaaaggact agcgcgcgag caagagaaaa tggtcgggcg cgcagttaat
tcatcgtgcg ctattactgt ttacaccccg gagccggagt actgggctgc ggggctgagg
ctcctcctcc tctttccccg gctccccact agcccccctc ccgagttccc aaagcagagg
gcggggaagc gagaggaggg aaaaaaatag agagaggtgg ggaagggaga aagagagatt
ctctggctaa tccccgccca cccgcccttt atattccggg ggtctgcgcg gccgaggacc
cctggctgcg ctgctctcag ctgccgggtc cgactcgcct cactcagctc ccctcctgcc
tcctgaaggg cagcttcgcc gacgcttggc gggaaaaaga agggagggga gggatcctga
gtcgcagtat aaaagaagct tttcgggcgt ttttttctga ctcgctgtag taattccagc
gagagacaga gggagtgagc ggacggttgg aagagccgtg tgtgcagagc cgcgctccgg
ggcgacctaa gaaggcagct ctggagtgag aggggctttg cctccgagcc tgccgcccac
tctccccaac cctgcgactg acccaacatc agcggccgca accctcgccg ccgctgggaa
actttgccca ttgcagcggg cagacacttc tcactggaac ttacaatctg cgagccagga
caggactccc caggctccgg ggagggaatt tttgtctatt tggggacagt gttctctgcc
tctgcccgcg atcagctctc ctgaaaagag ctcctcgagc tgtttgaagg ctggatttcc
tttgggcgtt ggaaaccccg gtaagcacag atctggtggt ctttccctgt gttctttctg
cgtcttgaat tgtagcggcc ggttaggaca gtctt

what part of this sequence are you interested in?

Nick

Here is the CG island sequence I got from Methprimer. It contains the promoter region.

acaccagccc agcccagcct attccagcct agctctgctc
atcccagctc attccagctc agcccagcct aacccagctc acaccagccc agcctattcc
agcctagctc tgctcatccc agctcattcc agctcagccc agcctaaccc aggtaacact
agtccagccc agcctattct agccaatccc agctcattcc agctcagctc agcctagccc
agctcacacc aggccaggcc cagcctattc cagcctagtt cagcccatcc cagctcattc
cgctcagctc agcctagccc agctcacacc agcccagccc agcccagcct attccagcct
agctcagctc agcccagccc agctcaggca gcctagcttg atagccagtt attgagccat
ccaaccctag tcagtcaggt cagcacagac catgccaatc tagtttagct agtccagtcc
agtttattcc agctcagcac aacccaccca gcccagccca gcctggccta gtttgggtag
cctagtctaa tttaacccca ttcagctgga gctcagctag tccattctac cctagcccag
accatgccag agtagctcag ctcatcccag ctcattatgg ttcatctcag ccaagcttag
ctcaggcagc ctagcttagc ctagcctatt ccagcctagc tcagtaagtc tagcctagct
catctcagct taatttagct tagcccagct cagtccactc atcccagatt agccacctgc
ggtgactgat atacgcaggg caagaacaca gttcagccga gcgctggcgc ccgaacaacc
gtacagaaag ggaaaggact agcgcgcgag caagagaaaa tggtcgggcg cgcagttaat
tcatcgtgcg ctattactgt ttacaccccg gagccggagt actgggctgc ggggctgagg
ctcctcctcc tctttccccg gctccccact agcccccctc ccgagttccc aaagcagagg
gcggggaagc gagaggaggg aaaaaaatag agagaggtgg ggaagggaga aagagagatt
ctctggctaa tccccgccca cccgcccttt atattccggg ggtctgcgcg gccgaggacc
cctggctgcg ctgctctcag ctgccgggtc cgactcgcct cactcagctc ccctcctgcc
tcctgaaggg cagcttcgcc gacgcttggc gggaaaaaga agggagggga gggatcctga
gtcgcagtat aaaagaagct tttcgggcgt ttttttctga ctcgctgtag taattccagc
gagagacaga gggagtgagc ggacggttgg aagagccgtg tgtgcagagc cgcgctccgg
ggcgacctaa gaaggcagct ctggagtgag aggggctttg cctccgagcc tgccgcccac
tctccccaac cctgcgactg acccaacatc agcggccgca accctcgccg ccgctgggaa
actttgccca ttgcagcggg cagacacttc tcactggaac ttacaatctg cgagccagga
caggactccc caggctccgg

I tried to use the Perlprimer to design BSP primers. Do I just simply paste the sequence? How do I set up the criteria?
It gave me too many primer pairs.

Thanks!

hi hn,

are you able to install perlprimer?

perlprimer requires activestate perl for windows before you can run it! I suggest you use perlprimer as it is able to pick more suitable BSP prikmers than methprimer.

All you need to do in perl primer is to goto the bisulfite PCR tab window, paste your sequence of interest and then hit the pick primers button and it will pick the primers for you. If you have any problems don't hesitate to ask.

I am on a computer that does not have perlprimer installed, I will get back to you on this one!

Nick

aagagagatt
ctctggctaa tccccgccca cccgcccttt atattccggg ggtctgcgcg gccgaggacc
cctggctgcg ctgctctcag ctgccgggtc cgactcgcct cactcagctc ccctcctgcc
tcctgaaggg cagcttcgcc gacgcttggc gggaaaaaga agggagggga gggatcctga
gtcgcagtat aaaagaagct tttcgggcgt ttttttctga ctcgctgtag

And this is the promoter region for this gene.

Thanks!