Calculation of Washing and Hybridization Stringencies
The Excel Spreadsheet at this link will allow calculation of washing stringencies from SSC concentration (e.g. 2x or 0.1x), formamide percentage (e.g. 40%) and temperature. If not, the filename stringen.xls at the end of the web address may work.
Here is a typical example
Stringency calculation |
|
|
|
%GC |
55 |
|
probe length |
20 |
(after labelling) |
formamide |
40 |
percentage |
SSC x |
2 |
in x of the 1 x SSC (20x SSC is 0.33 M Na) |
Hyb temp |
37 |
|
|
|
|
0.41gc |
22.55 |
|
[Na+] |
0.66 |
|
16.6LogNa |
-2.99557 |
|
500/l |
25 |
|
.61form |
24.4 |
|
|
1 |
mismatch factor (1 for >150bp. 5 for <20bp) |
Tm |
51.65443 |
|
|
|
|
String |
85.34557 |
|
Pat Heslop-Harrison/Trude Schwarzacher December 2003 |