Matt:LabNotes/2013-6-6: Difference between revisions
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<-- | <!-- ===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> | ||
4 tubes of 50ul with 50ul beads <br> | 4 tubes of 50ul with 50ul beads <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 <br> --> |
Revision as of 23:51, 5 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