Matt:LabNotes/2016-9-14: Difference between revisions
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>Mzcai m (→USER) |
>Mzcai m (→DpnII) |
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| align="center" style="background:#f0f0f0;"|'''Volume''' | | align="center" style="background:#f0f0f0;"|'''Volume''' | ||
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| 10X DpnII Buffer|| | | 10X DpnII Buffer||2 | ||
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| 100uM RE-DpnII_V6 guide oligo||5 | |||
|- | |||
| H2O||8 | |||
|- | |- | ||
|} | |} | ||
*Incubated at 94C for 2 min, then 37C for 3 min | *Incubated at 94C for 2 min, then 37C for 3 min | ||
*Added 5ul DpnII | *Added 5ul DpnII | ||
*Incubated at 37C for overnight ( | *Incubated at 37C for overnight (12 hrs) | ||
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===TBU Gel: Digestion Check=== | ===TBU Gel: Digestion Check=== |
Revision as of 16:50, 17 September 2016
CA12k_Nov2014 V6 (RevComp of V4) Probe Production
Production PCR
- V6 master mix
Components | Volume (1X) | Volume (50X) |
First round amplicon CA12k_Nov14_V6 (10nM) | 0.2 | 10 |
2X KAPA SYBG MM | 50 | 2500 |
AP1V6U (100uM) | 0.4 | 20 |
AP2V6 (100uM) | 0.4 | 20 |
H2O | 49 | 2450 |
Total | 100 | 5000 |
Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold
EtOH Precipitation
- 12 5-ml tubes (with 8 wells of PCR product each)
- 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 100ul H2O
Qia Column Purification
- Purified 12 tubes of 100ul in 12 columns following Qiagen protocol
- Eluted each column with 50ul and combined all V6 probes into one 1.5ml and all V8 probes into another
- Measured concentration with Nanodrop:
~600ul of V6 probes: 113.2 ng/ul => ~ 67.9 ug
Lambda Exonuclease Digestion
- Divide each into 8 pcr tubes of 75ul with total amplicon of <10ug each
Components | Volume |
Amplicon | 75 |
10X Lambda Exo Buffer | 10 |
Lambda Exonuclease | 10 |
H2O | 5 |
Total | 100 |
- Incubated at 37C for 1hr
- Purified with 8 Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- V6: 80.3ng/ul x 320ul = 25.7ug (75.6% yield)
Remove Amplification Adapters
USER
- Split each set into 5 PCR tubes and add 5ul USER
Components | Volume |
SS-amplicon | 63 |
USER | 5 |
DpnII 10X | 8 |
H2O | 4 |
Total | 80 |
- Incubate at 37C for 3 hours
DpnII
- Added 15ul of the following
Components | Volume |
10X DpnII Buffer | 2 |
100uM RE-DpnII_V6 guide oligo | 5 |
H2O | 8 |
- Incubated at 94C for 2 min, then 37C for 3 min
- Added 5ul DpnII
- Incubated at 37C for overnight (12 hrs)