Revision as of 21:25, 29 June 2013 by >Mzcai
Probe Capture
'
|
20 gap
|
0 gap
|
'
|
Probe Size |
12964 |
13179 |
|
gDNA HAPMAP C1 (87ng/ul) |
300 ng |
300 ng |
|
gDNA MW |
1.95x10^12 g/mol |
1.95x10^12 g/mol |
|
gDNA |
1.5385x10^-19 mol |
1.5385x10^-19 mol |
|
Probe Required (1000:1) |
1.5385x10^-16 mol |
1.5385x10^-16 mol |
|
Probe MW |
8.1419x10^8 g/mol |
8.2769x10^8 g/mol |
(Probe size x (193nt x 325 Da/nt + 79Da)
|
Amount Probe Required |
125 ng (7.44ul) |
127 ng (4.47 ul) |
|
|
0 gap probes from 2013-6-27
20 gap probes from 2013-6-6
cDNA from 2013-6-26
Tube #
|
Probe type
|
Samples
|
Probes (ul)
|
10X AmpLigase Buffer (ul)
|
Target (ul)
|
H2O (ul)
|
Total
|
1 |
0 gap (28.4ng/ul) |
NTC |
4.47 |
3 |
0 |
22.53 |
30
|
2 |
0 gap (28.4ng/ul) |
gDNA HAPMAP C1 (87ng/ul) |
4.47 |
3 |
3.45 |
19.08 |
30
|
3 |
0 gap (28.4ng/ul) |
cDNA |
4.47 |
3 |
20 |
2.53 |
30
|
4 |
20 gap (16.79ng/ul) |
NTC |
7.44 |
3 |
0 |
19.56 |
30
|
5 |
20 gap (16.79ng/ul) |
gDNA HAPMAP C1 (87ng/ul) |
7.44 |
3 |
3.45 |
16.11 |
30
|
6 |
20 gap (16.79ng/ul) |
cDNA |
7.44 |
3 |
20 |
0 |
30.44
|
|
Program
- 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 20h
- To 0 gap probes -> add 3ul AmpLigase enzyme mix (0.5U/ul AmpLigase in 1X AmpLigase buffer)
- To 20 gap probes -> add 2ul SLN mix (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP mix)
- -> 55 C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
AmpLigase enzyme mix
Components
|
Stock conc.
|
Unit
|
Final conc.
|
Unit
|
Prepare volume 10ul
|
AmpLigase |
5 |
U/ul |
0.5 |
U/ul |
1.00
|
10x AmpLigase Buffer |
10 |
x |
1 |
x |
1.00
|
H2O |
|
|
|
|
8.00
|
Total |
|
|
|
|
10.00
|
SLN mix
Components
|
Stock conc.
|
Unit
|
Final conc.
|
Unit
|
Prepare volume 10ul
|
AmpLigase |
5 |
U/ul |
0.5 |
U/ul |
1.00
|
10x AmpLigase Buffer |
10 |
x |
1 |
x |
1.00
|
dNTP Mix |
1 |
mM |
100 |
uM |
1.00
|
Stoffel |
10 |
U/ul |
2 |
U/ul |
2.00
|
H2O |
|
|
|
|
5.00
|
Total |
|
|
|
|
10.00
|