Location via proxy:   [ UP ]  
[Report a bug]   [Manage cookies]                
Scribd
Upload
13 views

Calculating TM Example

This document describes how to calculate the melting temperature (Tm) of an oligonucleotide using the nearest neighbor method. It provides the equations and thermodynamic parameters needed to calculate Tm at different sodium ion concentrations. For the oligonucleotide 5'-CGTTGA-3', the document calculates that: (a) at [Na+] = 1M, Tm = 66.36°C (b) at [Na+] = 250mM, Tm = 59.37°C (c) at [Na+] = 75mM, Tm = 51.46°C.

Uploaded by

Sarah Khaze
Copyright
© Attribution Non-Commercial (BY-NC)
Available Formats
Download as PDF, TXT or read online on Scribd
13 views

Calculating TM Example

This document describes how to calculate the melting temperature (Tm) of an oligonucleotide using the nearest neighbor method. It provides the equations and thermodynamic parameters needed to calculate Tm at different sodium ion concentrations. For the oligonucleotide 5'-CGTTGA-3', the document calculates that: (a) at [Na+] = 1M, Tm = 66.36°C (b) at [Na+] = 250mM, Tm = 59.37°C (c) at [Na+] = 75mM, Tm = 51.46°C.

Uploaded by

Sarah Khaze
Copyright
© Attribution Non-Commercial (BY-NC)
Available Formats
Download as PDF, TXT or read online on Scribd
You are on page 1/ 2

Determining Tm Using the Nearest Neighbor Method

Example is determine to the Tm of the following oligonucleotide:


5-CGTTGA-3
3-GCAACT-5
What is Tm when:
CT = 50 mM and (a) [Na+] = 1M (b) [Na+] = 250 mM or (c) [Na+] = 75 mM?
So, looking at the table, we get the following:
-1

Terminal C/G
CG/GC
GT/CA
TT/AA
TG/AC
GA/CT
Terminal A/T

-1

H (cal mol )
100
-10600
-8400
-7900
-8500
-8200
2300

-1

S (cal mol K )
-2.8
-27.2
-22.4
-22.2
-22.7
-22.2
4.1

H = H (terminal G/C) + H (CG/GC) + H (GT/CA) + H (TT/AA) +


H (TG/AC) + H(GA/CT) + H (terminal A/T)
H = 100 + -10600 + -8400 + -7900 + -8500 + -8200 + 2300
-1

H = -41,200 cal mol

S = -2.8 +(-27.2) + (-22.4) + (-22.2) + (-22.7) + (-22.2) + (4.1)


-1

-1

S = -115.4 cal mol K

Tm =

H
S + R ln CT

S = S[Na+]=1M + 0.368(N-1)ln[Na+]
& R = 1.9872 cal mol-1 K-1

(a) CT = 50 mM and [Na+] = 1M


-1

-1

Tm = -41,200 cal mol-1 / (-115.4 cal mol K ) + (1.9872 cal mol-1 K-1)(ln 0.05M)
-1

-1

Tm = -41,200 cal mol-1 / (-115.4 cal mol K ) + (-5.953 cal mol-1 K-1)
-1

-1

Tm = -41,200 cal mol-1 / -121.353 cal mol K


Tm = 339.51 K = 66.36 C
(b) CT = 50 mM and [Na+] = 250 mM
S = S[Na+]=1M + 0.368(N-1)ln[Na+]
-1

-1

S = (-115.4 cal mol K ) + 0.368(6-1)ln(0.25 M)


-1

-1

-1

-1

S = (-115.4 cal mol K ) + (-2.551 cal mol K )


-1

-1

S = -117.95 cal mol K


Therefore
-1

-1

Tm = -41,200 cal mol-1 / (-117.95 cal mol K ) + (-5.953 cal mol-1 K-1)
-1

-1

Tm = -41,200 cal mol-1 / -123.903 cal mol K


Tm = 332.52 K = 59.37 C
(b) CT = 50 mM and [Na+] = 75 mM
S = S[Na+]=1M + 0.368(N-1)ln[Na+]
-1

-1

S = (-115.4 cal mol K ) + 0.368(6-1)ln(0.075 M)


-1

-1

-1

-1

S = (-115.4 cal mol K ) + (-4.766 cal mol K )


-1

-1

S = -120.17 cal mol K


Therefore
-1

-1

Tm = -41,200 cal mol-1 / (-120.97 cal mol K ) + (-5.953 cal mol-1 K-1)
-1

-1

Tm = -41,200 cal mol-1 / -126.923 cal mol K


Tm = 324.61 K = 51.46 C

You might also like