calculating Tm with formamide

Donald W. Back nstn2072 at fox.nstn.ca
Sun Jun 18 06:56:42 EST 1995


In article <jpcd0-0806951402130001 at macr1-6.welc.cam.ac.uk> jpcd0 at mole.bio.cam.ac.uk (John Dixon) writes:
>From: jpcd0 at mole.bio.cam.ac.uk (John Dixon)
>Subject: calculating Tm with formamide
>Date: Thu, 08 Jun 1995 14:02:13 +0000

>Hi, can anyone let me know where I can find an equation for calculating
>the Tm of primers when the buffer is supplemented with varying percentages
>of formamide?
>No luck in Maniatis or practical approach.

>Cheers, Johnny D

>-- 
>John Dixon                     Lab 44 (223) 334131
>Wellcome/CRC Institute         Fax 44 (223) 334134
>Dept Genetics
>Cambridge University    
>United Kingdom       e-m: jpcd0 at mole.bio.cam.ac.uk


A. DNA probes on DNA blots:

Tm  = 81.5 + (16.6 log M) + 41(mole fraction G+C) - (500/L) - 0.62 (% formamide)

Tm = melting temperature
M = monovalent cation concentration, [Na+] in moles/litre
L = length of probe molecule, a minor consideration in most cases


B. RNA probes on DNA blots:

-The equation compensates for the increased stability of RNA/DNA hybrids.

Tm = 79.8 + (18.5 log(M)) + 58.4(mole fraction G+C) + 11.8(mole fraction G+C)2 - (820/L) - 0.5(% formamide)

C. RNA probes on RNA blots:

-RNA probes on RNA blots present a major problem because of the very abundant, 
 G/C rich (rat = 65%G/C) 18S and 28S ribosomal RNAs. Failure to hybridize 
 under stringent enough conditions will result in very significant 
 non-specific hybridization to ribosomal RNAs.

-Equation compensates for the increased stability of RNA/RNA hybrids:

Tm = 79.8 + 18.5 log M + 58.4(mole fraction G/C) + 11.8(mole fraction G/C)2 -820/L - 0.35(%formamide)


Enjoy,

Don Back


Donald W. Back. Ph.D.
15 Regency Court
Kingston, Ontario. K7K 6G1
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E-Mail: nstn2072 at fox.nstn.ca



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