Matt:LabNotes/2014-9-18: Difference between revisions
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Revision as of 22:18, 23 September 2014
Agi26k0gap Probe Production
Production PCR
- Make Master Mix for 51 well (7 strips)
- 0 gap first round amplicon -> V6 primers
Components | 1x rxn (ul) | 50x rxn (ul) |
First round amplicon (10nM) | 0.2 | 10 |
2x Kapa SYBR MM | 50 | 2500 |
100uM pAP1V6U | 0.4 | 20 |
100uM AP2V6 | 0.4 | 20 |
H2O | 49 | 2450 |
Total | 100 | 5000 |
98C 30sec -> (98C 10sec -> 55C 20sec -> 72C 30sec) x 15 -> 72C 2min -> 15C hold
File:20140919 Agi26k0gapProductionPCR.PNG
EtOH Precipitation
- 6 5-ml tubes (with 8.5 wells of PCR product each) for 0gap
- 850ul PCR product
- 2000ul 100% EtOH
- 2.7ul GlycoBlue
- 85ul 3M NaOAc pH 5.2-5.5
- Vortexed and put in -80C for overnight
- Centrifuged at 3000rpm at 4C for 30min
- Discarded supernatant and added 800ul of cold 80% EtOH
- Transferred DNA pellet (with 1ml pipette tip) to 6 1.5mL tubes
- Centrifuged 1.5ml tubes at 15,000rpm for 5 min on table-top centrifuge
- Discarded supernatant and air-dried for 5 min in hood
- Resuspended DNA with 100ul H2O
Qia Column Purification
- Purified 6 tubes of 100ul in 6 columns following Qiagen protocol
- Eluted each column with 40ul and combined all probes into one 1.5ml
- Measured concentration with Nanodrop:
- ~225ul of Agi26k0gap probes: 335.3 ng/ul => 75.4 ug
Lambda Exonuclease Digestion
- Divide into eight pcr tubes of 29ul with total amplicon of ~10ug each
Components | Volume |
Amplicon | 29 |
10X Lambda Exo Buffer | 10 |
Lambda Exonuclease | 10 |
H2O | 51 |
Total | 100 |
- Incubated at 37C for 1hr
- Purified with Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 67.8ng/ul x 310ul = 21.0ug
Removal of Amplification Adapters
Components | Volume |
ss-amplicon | 62 |
USER | 5 |
H2O | 13 |
total | 80 |
*~5ug ssDNA per tube
- Incubated at 37C for 2 hours