Matt:LabNotes/2013-6-28
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Probe Capture[edit]
' | 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.
- No Exo I/III mix was added to NTC samples because lack of Exo I enzyme
Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-7-1
AmpLigase enzyme mix[edit]
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[edit]
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 |