Matt:LabNotes/2014-8-15
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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:2014-08-14 Agi26k0gapProductionPCR.JPG
EtOH Precipitation
- 6 15-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: 317.5 ng/ul => 71.4 ug
Lambda Exonuclease Digestion
- Divide into seven pcr tubes of 32ul with total amplicon of ~10ug each
- Seventh tube had 33ul of amplicon
Components | Volume |
Amplicon | 32 |
10X Lambda Exo Buffer | 10 |
Lambda Exonuclease | 10 |
H2O | 48 |
Total | 100 |
- Incubated at 37C for 1hr
- Purified with Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 89.0ng/ul x 255ul = 22.7ug (63.6% yield)
Removal of Amplification Adapters
Components | Volume |
ss-amplicon | 63.5 |
USER | 5 |
H2O | 11.5 |
total | 80 |
*~5ug ssDNA per tube
- Incubated at 37C for 2 hours
- Added 15ul of the following
Components | Volume |
10X DpnII Buffer | 10 |
100uM RE-DpnII guide oligo | 5 |
RE-DpnII guide oligo: 0-gap: RE DpnII V6
- Incubated at 94C for 2 min, then 37C for 3 min
- Added 5ul DpnII
- Incubated at 37C for 3 hr
- Added 100ul of the following
Components | Volume |
10X DpnII Buffer | 10 |
USER | 5 |
H2O | 85 |
Total | 100 |
- Incubated at 37C for overnight (~15 hrs)
TBU Gel Check
- Combine the 4 tubes
- 2ul Sample + 2ul 2X Urea Loading Dye
- 0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye
- Digested sample
- Undigested sample: 10nM 1st round amplicon
File:2014-08-17 Agi26k0gapDigestCheck.jpg
- Brightest band below 200bp as expected
Column Purification
- Used Zymo ssDNA/RNA columns
- Total Eluted: 260 ul
- Nanodrop quant: 50.8 ng/ul
- Yield: 50.8 ng/ul x 260 ul = 13.2 ug