PCR amplificaiton isnt working - (Nov/11/2007 )
QUOTE (RandomGuy187 @ Nov 13 2007, 02:48 AM)
QUOTE (JessH @ Nov 11 2007, 11:04 PM)
If you are going to re-design your primers you could consider making some of the bases LNA (Locked Nucleic Acid) bases. These are analogues of nucleotides, which increases the strength of the bonds and therefore the melting temperature. I first heard about them at a qPCR users meeting which I think was run by Stratagene? My memory is not best and I could only find information about them on the Sigma website:
http://www.sigmaaldrich.com/Brands/Sigma_G...LNA_Oligos.html
This journal may provide you with more information:
http://nar.oxfordjournals.org/cgi/content/full/gkl756v1
Cheers, Jess
http://www.sigmaaldrich.com/Brands/Sigma_G...LNA_Oligos.html
This journal may provide you with more information:
http://nar.oxfordjournals.org/cgi/content/full/gkl756v1
Cheers, Jess
Ill look into this, thanks.
My boss just checked out the price of LNAs and they're not cheap. We pay $AUD0.95 per standard desalted base here and primers with LNAs are a whopping $AUD39.00 per LNA base! Before you totally disregard them though, you only need two or three at the 5` end of the primer, so it's not cheap but still worth exploring if you're really struggling and willing to fork out the cash.
-killerkoz17-
Lower extension temperature prevents the 3' end of the primed extension region from failing to extend, due to poor binding of the AT rich region to the template strand. With a 72C extension, a highly AT rich region of 25-30 bp will prevent any extension of the product. See Su XZ, Wu Y, Sifri CD, Wellems TE.
Reduced extension temperatures required for PCR amplification of extremely A+T-rich DNA.
Nucleic Acids Res. 1996 Apr 15;24(8):1574-5. PMID: 8628694
-phage434-
QUOTE (killerkoz17 @ Nov 13 2007, 04:06 PM)
QUOTE (RandomGuy187 @ Nov 13 2007, 02:48 AM)
QUOTE (JessH @ Nov 11 2007, 11:04 PM)
If you are going to re-design your primers you could consider making some of the bases LNA (Locked Nucleic Acid) bases. These are analogues of nucleotides, which increases the strength of the bonds and therefore the melting temperature. I first heard about them at a qPCR users meeting which I think was run by Stratagene? My memory is not best and I could only find information about them on the Sigma website:
http://www.sigmaaldrich.com/Brands/Sigma_G...LNA_Oligos.html
This journal may provide you with more information:
http://nar.oxfordjournals.org/cgi/content/full/gkl756v1
Cheers, Jess
http://www.sigmaaldrich.com/Brands/Sigma_G...LNA_Oligos.html
This journal may provide you with more information:
http://nar.oxfordjournals.org/cgi/content/full/gkl756v1
Cheers, Jess
Ill look into this, thanks.
My boss just checked out the price of LNAs and they're not cheap. We pay $AUD0.95 per standard desalted base here and primers with LNAs are a whopping $AUD39.00 per LNA base! Before you totally disregard them though, you only need two or three at the 5` end of the primer, so it's not cheap but still worth exploring if you're really struggling and willing to fork out the cash.
OUCH!
I wasn't really sure of the price.
The Phusion polymerase and PCR in 2-steps are really good suggestions.
-JessH-