Matt:LabNotes/2016-11-10: Difference between revisions
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**120ul 3M NaOAc pH 5.2-5.5 | **120ul 3M NaOAc pH 5.2-5.5 | ||
*Vortexed and put in -80C for | *Vortexed and put in -80C for overnight | ||
*Centrifuged at 3000rpm at 4C for 30 min | *Centrifuged at 3000rpm at 4C for 30 min | ||
*Discarded supernatant and added 800ul of cold 80% EtOH | *Discarded supernatant and added 800ul of cold 80% EtOH | ||
*Transferred DNA pellet (with 1ml pipette tip) to | *Transferred DNA pellet (with 1ml pipette tip) to 16 1.5mL tubes | ||
*Centrifuged 1.5ml tubes at 14,000rpm for 5 min at 4C | *Centrifuged 1.5ml tubes at 14,000rpm for 5 min at 4C | ||
*Discarded supernatant and air-dried for 5 min in hood | *Discarded supernatant and air-dried for 5 min in hood |
Revision as of 23:23, 12 November 2016
CA12k_Oct2016 V4 Probe Preparation
Production PCR
- V4 master mix
Components | Volume (1X) | Volume (50X) |
First round amplicon CA12k_Oct2016_V4 (10nM) | 0.2 | 10 |
2X KAPA SYBG MM | 50 | 2500 |
AP1V4U (100uM) | 0.4 | 20 |
AP2V4 (100uM) | 0.4 | 20 |
H2O | 49 | 2450 |
Total | 100 | 5000 |
- Did one plate - 96 wells 100ul each
Program 98C 1min -> (98C 30sec -> 55C 45sec -> 72C 45sec) x 14 -> 72C 2min -> 15C hold 450px
EtOH Precipitation
- 16 5-ml tubes each with
- 1,200ul PCR product
- 3000ul 100% EtOH
- 4ul GlycoBlue
- 120ul 3M NaOAc pH 5.2-5.5
- Vortexed and put in -80C for overnight
- Centrifuged at 3000rpm at 4C for 30 min
- Discarded supernatant and added 800ul of cold 80% EtOH
- Transferred DNA pellet (with 1ml pipette tip) to 16 1.5mL tubes
- Centrifuged 1.5ml tubes at 14,000rpm for 5 min at 4C
- Discarded supernatant and air-dried for 5 min in hood
- Resuspended DNA with 100ul H2O
Qia Column Purification
- 8 columns elute 50ul each