Matt:LabNotes/2014-7-17
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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:2014-07-17 Agi26k0gapProductionPCR.JPG
EtOH Precipitation[edit]
- 6 15-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 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[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: 352.2 ng/ul => 77.5 ug
- Every time I tried to measure sample it gave me a warning message that there might be a bubble or unclean pedastal, but consistently measured ~355ng/ul (all 3 times)
- In between the 3 sample measurements I cleaned and measured a blank water sample and got +/-2ng/ul with no warning message
- Next day re-measured and got 355.5 ng/ul with no error
Lambda Exonuclease Digestion[edit]
- Divide into eight pcr tubes of 27.5ul with total amplicon of ~10ug each
- Four with old Lambda and Four with new Lambda
- Fourth tube of each set had extra 2.5ul amplicon
- Four with old Lambda and Four with new Lambda
Components | Volume |
Amplicon | 27.5 |
10X Lambda Exo Buffer | 10 |
Lambda Exonuclease | 10 |
H2O | 52.5 |
Total | 100 |
- Incubated at 37C for 1hr
- Purified with of Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Combined the three tubes with 27.5ul amplicon to measure and compare
- Recombined and measured ssDNA with Nanodrop:
- New Lambda: 69.5ng/ul x 120ul = 8.34ug
- Old Lambda: 65.3ng/ul x 120ul = 7.84ug
- Estimate total yield: ~67ng/ul x 320ul = 21.44ug (21.44/38.75 = 55.3%)
- Result: Negligible difference between old and new lambda
- Not sure what was causing low yield during Lambda digestion Matt:LabNotes/2014-6-24
- Checked Qiagen vs Zymo columns and that wasn't the reason either
- Whatever it was, things seem to work well now
- Continued on: Matt:LabNotes/2014-7-22