Matt:LabNotes/2016-9-14: Difference between revisions
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>Mzcai |
>Mzcai m (→USER) |
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| align="center" style="background:#f0f0f0;"|'''Volume''' | | align="center" style="background:#f0f0f0;"|'''Volume''' | ||
|- | |- | ||
| SS-amplicon|| | | SS-amplicon||63 | ||
|- | |- | ||
| USER||5 | | USER||5 | ||
|- | |||
| DpnII 10X||8 | |||
|- | |||
| H2O||4 | |||
|- | |- | ||
| Total||80 | | Total||80 | ||
|} | |} | ||
*Incubate at 37C for 3 hours | *Incubate at 37C for 3 hours | ||
====DpnII==== | ====DpnII==== | ||
*Added 15ul of the following | *Added 15ul of the following |
Revision as of 01:04, 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 | 10 |
100uM RE-DpnII guide oligo | 5 |
RE-DpnII guide oligo: V6: RE DpnII V6
- Incubated at 94C for 2 min, then 37C for 3 min
- Added 5ul DpnII
- Incubated at 37C for overnight (16 hrs)
TBU Gel: Digestion Check
- Combine into 1 tube for each set
- 2ul Digested Sample + 2ul 2X Urea Loading Dye
- 5ul Undigested sample (10nM V8 1st round amplicon) + 5ul 2X Urea Loading Dye
- 0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye
[[File:]]
Zymo Column Purification
- 3 columns for each set
- Eluted 20ul each column (60ul total)
- Nanodrop
- V6: 137.3ng/ul x 60ul = 8.2ug
PAGE Size Selection
- Run 4 gels for V6
- 200V for 35min
Components | 2X Volume |
V6 Probes | 60 |
H2O | 60 |
TBE-Urea Buffer 2X | 120 |
Components | Volume |
Low Mass Ladder | 4 |
TBE-Urea Buffer 2X | 20 |
H2O | 16 |
Total | 40 |
EtOH Precipitation
- Put cut out gel in 0.5mL tube (with holes at bottom and centrifuged the tube at 15,000 rpm, for 3 min at RT.
- Transfered the gel remaining in 0.5 mL tube to 2.0 mL tube below with pipette tip.
- Added 900 ul of 1X TE buffer
- Vortexed for 60 min at 37 C in incubator
- Centrifuged at 15,000 rpm for 3 min at RT
- Transfered the clear spnt. to 8 Nanosep column and centrifuged at 15,000 rpm for 3 min
- Transfered the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min
- Transfered spnt to fresh 1.5 mL tube (~400ul per tube)
- Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 40ul of 3M NaoAc pH 5.2
- Vortexed and placed the 8 tubes at -80C for 30 min
- Spun 8 tubes at 10,000rpm at 4C for 30min
- Discard supernatant and add 650ul 80% EtOH and spin at 14,000rpm at 4C for 5min
- Discard supernatant and let dry in hood for 10min
- Resuspend each tube with 10ul and recombine (40ul total each)
File:2015-01-30 V6 GelQuant.jpg
- V6: 41.7ng/ul => ng/ul / (150nt*325Da/nt + 79Da) = 854nM (38ul)