Matt:LabNotes/2014-5-22
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Agi26k0gap Probe Production[edit]
- Reference: Matt:LabNotes/2013-5-10
- Use 20nM aliquot of Agilent oligos in tube labeled "FISHYB V1 probes 5/07/13"
Probe Expansion PCR[edit]
- Expansion PCR in 200ul total: 2 tubes of 100ul for 0bp gap probes with AP1V6 primers
' | 0 gap probes (ul) |
Agi26k (20nM) | 10 |
2x Kapa SYBG qPCR MM | 100 |
100uM pAP1V6U | 0.8 |
100uM AP2V6 | 0.8 |
H2O | 88.4 |
Total | 200 |
- Mixed thoroughly and then split into two tubes
94C 2min -> (94C 30sec -> 54C 45sec -> 72C 45sec) x 12 -> 72C 3min -> 15C hold
File:20140522 Agi26k0gapExpansionPCR.JPG
- Purified with 2 Qiagen columns
- Eluted with 40ul each tube and combined into two tubes (80ul total)
- Nanodrop: 32.2 ng/ul -> 32.2ng/ul / (228bp*660Da/bp) = 214nM
- Dilute 2.34ul in 47.66ul H2O
- Final volumes:
- 75ul of 214nM
- 50ul of 10nM
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:052314 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 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 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: 295.8 ng/ul => 65.1ug
Lambda Exonuclease Digestion[edit]
- Divide each tube of 220ul into two 1.5ml tubes of 110ul with total amplicon of ~30ug
Components | Volume (ul) |
Amplicon | 110 |
10X Lambda Exo Buffer | 15 |
Lambda Exonuclease | 10 |
H2O | 15 |
total | 150 |
- Incubated at 37C for 1hr
- Purified each of the tubes with 3 columns (6 columns total) of Zymo ssDNA/RNA kit
- Eluted with 27ul each
- Recombined and measured ssDNA with Nanodrop:
- 49.2ng/ul x 160ul = 7.87ug (12% yield)
- Why is it so low? Expected to be about 40%...
- Low yield could be because Zymo kit is for ssDNA between 17nt-200nt and these oligos are slightly above 200nt before USER & DpnII digestion
- 49.2ng/ul x 160ul = 7.87ug (12% yield)
- Noi suggests I may have had too much DNA in each tube for lambda digestion. She says 10ug DNA for 100ul reaction (15ug/150ul reaction)
- Continued on: Matt:LabNotes/2014-6-24