Matt:LabNotes/2013-6-6: Difference between revisions
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* Did same number of PCR cycles as [http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-5-11 Production PCR] to keep amplification as consistent as possible | * Did same number of PCR cycles as [http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-5-11 Production PCR] to keep amplification as consistent as possible | ||
2 reactions of Dan's V6 probes: | |||
{| {{table}} border = 1 | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume''' | |||
|- | |||
| 10nM Agilent Expansion PCRd 0 gap Oligos||0.20 | |||
|- | |||
| 100uM Forward ISB_AP1V6_AFv2||0.40 | |||
|- | |||
| 100uM Reverse ISB_AP2V6_AR||0.40 | |||
|- | |||
| 2x KAPA SYBG fast MM||50 | |||
|- | |||
| H2O||49 | |||
|- | |||
| Total||100 | |||
|} | |||
2 reactions of Dan's V4 probes: | |||
{| {{table}} border = 1 | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume''' | |||
|- | |||
| 10nM Agilent Expansion PCRd 20 gap Oligos||0.20 | |||
|- | |||
| 100uM Forward ISB_AP1V4_AFv2||0.40 | |||
|- | |||
| 100uM Reverse ISB_AP2V4_AR||0.40 | |||
|- | |||
| 2x KAPA SYBG fast MM||50 | |||
|- | |||
| H2O||49 | |||
|- | |||
| Total||100 | |||
|} | |||
'''Dan's Probes Program''' | |||
98C 30s -> (98C 5s -> 50C 20s -> 72C 30s)x5 -> (98C 5s -> 72C 30s)x8 -> 72C 3min | |||
*13 cycles total | |||
*First made 9X Master Mix of KAPA and H2O: 450ul KAPA + 441ul H2O | |||
<!-- ===Bead Purify + Measure Conc=== | <!-- ===Bead Purify + Measure Conc=== | ||
Following: [http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/Bead_Purification_Protocol|Bead Purification Protocol] <br> | Following: [http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/Bead_Purification_Protocol|Bead Purification Protocol] <br> | ||
16 tubes of 50ul with 50ul beads (2 tubes for each PCR)<br> | |||
Elute with 12.5ul H20 in each tube to make 50ul total <br> --> | Elute with 12.5ul H20 in each tube to make 50ul total for each sample<br> --> |
Revision as of 17:18, 6 June 2013
Sequence Agilent Oligos
PCR
PCR Mix
2 reactions of 0 gap probes:
Components | Volume |
10nM Agilent Expansion PCRd 0 gap Oligos | 0.20 |
100uM Forward ISB_AP1V6_AFv1 | 0.40 |
100uM Reverse ISB_AP2V6_AR | 0.40 |
2x KAPA SYBG fast MM | 50 |
H2O | 49 |
Total | 100 |
2 reactions of 20 gap probes:
Components | Volume |
10nM Agilent Expansion PCRd 20 gap Oligos | 0.20 |
100uM Forward ISB_AP1V4_AFv1 | 0.40 |
100uM Reverse ISB_AP2V4_AR | 0.40 |
2x KAPA SYBG fast MM | 50 |
H2O | 49 |
Total | 100 |
Agilent Oligos: Expansion PCRd but before Production PCR (see: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-5-10)
Program 98C 30s -> (98C 5s -> 50C 20s -> 72C 30s)x5 -> (98C 5s -> 72C 30s)x10 -> 72C 3min
- 2 tubes of each in case concentration is low
- Did same number of PCR cycles as Production PCR to keep amplification as consistent as possible
2 reactions of Dan's V6 probes:
Components | Volume |
10nM Agilent Expansion PCRd 0 gap Oligos | 0.20 |
100uM Forward ISB_AP1V6_AFv2 | 0.40 |
100uM Reverse ISB_AP2V6_AR | 0.40 |
2x KAPA SYBG fast MM | 50 |
H2O | 49 |
Total | 100 |
2 reactions of Dan's V4 probes:
Components | Volume |
10nM Agilent Expansion PCRd 20 gap Oligos | 0.20 |
100uM Forward ISB_AP1V4_AFv2 | 0.40 |
100uM Reverse ISB_AP2V4_AR | 0.40 |
2x KAPA SYBG fast MM | 50 |
H2O | 49 |
Total | 100 |
Dan's Probes Program 98C 30s -> (98C 5s -> 50C 20s -> 72C 30s)x5 -> (98C 5s -> 72C 30s)x8 -> 72C 3min
- 13 cycles total
*First made 9X Master Mix of KAPA and H2O: 450ul KAPA + 441ul H2O