Matt:LabNotes/2015-1-15: Difference between revisions
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*5ul Undigested V7 10nM 1st round amplicon) + 5ul 2X Urea Loading Dye | *5ul Undigested V7 10nM 1st round amplicon) + 5ul 2X Urea Loading Dye | ||
*2X (0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye) | *2X (0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye) | ||
[[File:2015-01-20_V4V7_Digest_GelCheck.jpg]] | |||
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===Zymo Column Purification=== | ===Zymo Column Purification=== |
Revision as of 21:16, 20 January 2015
CA12k_Nov2014 V4 and V7 Probe Production
- Repeat Matt:LabNotes/2015-1-5
Production PCR
- V4 master mix
Components | Volume (1X) | Volume (50X) |
First round amplicon CA12k_Nov14_V4 (10nM) | 0.2 | 10 |
2X KAPA SYBG MM | 50 | 2500 |
AP1V4U (100uM) | 0.4 | 20 |
AP2V4 (100uM) | 0.4 | 20 |
H2O | 49 | 2450 |
Total | 100 | 5000 |
- V7 master mix
Components | Volume (1X) | Volume (50X) |
First round amplicon CA12k_Nov14_V7 (10nM) | 0.2 | 10 |
2X KAPA SYBG MM | 50 | 2500 |
AP1V7U (100uM) | 0.4 | 20 |
AP2V7 (100uM) | 0.4 | 20 |
H2O | 49 | 2450 |
Total | 100 | 5000 |
Program 98C 1min -> (98C 15sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold [[File: | 800px]]
EtOH Precipitation
- 6 15-ml tubes (with 8 wells of PCR product each) for V4 and 6 tubes for V7
- 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 12 1.5mL tubes
- Centrifuged 1.5ml tubes at 14,000rpm for 5 min at 4C
- Discarded supernatant and air-dried for 5 min in hood
- Resuspended DNA with 50ul H2O
Qia Column Purification
- Purified in 6 columns following Qiagen protocol
- Eluted each column with 50ul and combined all V4 probes into one 1.5ml and all V7 probes into another
- Measured concentration with Nanodrop:
~150ul of V4 probes: 156.5 ng/ul => ~23.5 ug ~150ul of V7 probes: 163.6 ng/ul => ~24.5 ug
Lambda Exonuclease Digestion
- Divide each into 3 pcr tubes of 50ul with total amplicon of <10ug each
Components | Volume |
Amplicon | 50 |
10X Lambda Exo Buffer | 10 |
Lambda Exonuclease | 10 |
H2O | 30 |
Total | 100 |
- Incubated at 37C for 1hr
- Purified with 3 Zymo ssDNA/RNA columns each
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- V4: 77.0ng/ul x 120ul = 9.24ug (78.6% yield)
- V7: 61.0ng/ul x 120ul = 7.32ug (59.8% yield)
Remove Amplification Adapters
USER
- Split each set into 2 PCR tubes and add 5ul USER
Components | Volume |
SS-amplicon | 60 |
USER | 5 |
H2O | 20 |
Total | 85 |
- Incubate at 37C for 2.5 hours
DpnII
- Added 15ul of the following
Components | Volume |
10X DpnII Buffer | 10 |
100uM RE-DpnII guide oligo | 5 |
RE-DpnII guide oligo: V4: RE DpnII V4 V7: RE DpnII V7
- Incubated at 94C for 2 min, then 37C for 3 min
- Added 5ul DpnII
- Incubated at 37C for overnight
TBU Gel: Digestion Check
- Combine into 1 tube for each set
- 2ul Digested Sample + 2ul 2X Urea Loading Dye
- 5ul Undigested V7 10nM 1st round amplicon) + 5ul 2X Urea Loading Dye
- 2X (0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye)