Matt:LabNotes/2014-7-1
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Agi26k0gap Probe Production[edit]
Production PCR[edit]
- Make Master Mix for 46 well (6 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:2014-07-2 Agi26k0gapProductionPCR.JPG
EtOH Precipitation[edit]
- 6 5-ml tubes (with 8 wells of PCR product each) for 0gap
- 800ul PCR product
- 2000ul 100% EtOH
- 2.7ul GlycoBlue
- 80ul 3M NaOAc pH 5.2-5.5
- Vortexed and put in -80C for 1hr
- 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 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[edit]
- 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:
~220ul of Agi26k0gap probes: 138.8 ng/ul => 33.3 ug
Lambda Exonuclease Digestion[edit]
- Divide into four pcr tubes of 55ul with total amplicon of ~10ug
Components | Volume |
Amplicon | 55 |
10X Lambda Exo Buffer | 10 |
Lambda Exonuclease | 10 |
H2O | 25 |
Total | 100 |
- Incubated at 37C for 1hr
- Purified 2 of the tubes with of Zymo ssDNA/RNA columns and 2 of the tubes with Qiagen PCR columns
- Eluted with 50ul each
- Recombined and measured ssDNA with Nanodrop:
- Zymo: 80.3 ng/ul x 100ul = ~8ug (8ug/16.65ug = 48% yield)
- Qiagen: 61.9 ng/ul x 100ul = ~6.2ug (6.2ug/16.65ug = 37% yield)
- Zymo is more efficient and this time had no trouble with efficiency
- Used new Lambda enzyme and buffer, maybe that is why it had higher yield
- Continued on: Matt:LabNotes/2014-7-22