Matt:LabNotes/2016-7-26: Difference between revisions
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>Mzcai (Created page with "=TB12k_Apr2016 V4 Probe Preparation= * 10nM Seed Oligos of TB12k_Apr2016 V4 ===Production PCR=== *Make 10ml V4 master mix for 96 wells {| {{table...") |
>Mzcai |
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Line 30: | Line 30: | ||
**4ul GlycoBlue | **4ul GlycoBlue | ||
**120ul 3M NaOAc pH 5.2-5.5 | **120ul 3M NaOAc pH 5.2-5.5 | ||
*Vortexed and put in -80C for | *Vortexed and put in -80C for 30 min | ||
*Centrifuged at 3000rpm at 4C for 30 min | *Centrifuged at 3000rpm at 4C for 30 min | ||
*Discarded supernatant and added 800ul of cold 80% EtOH | *Discarded supernatant and added 800ul of cold 80% EtOH |
Revision as of 22:20, 26 July 2016
TB12k_Apr2016 V4 Probe Preparation
Production PCR
- Make 10ml V4 master mix for 96 wells
Components | Volume (1X) | Volume (100X) |
First round amplicon TB12k_Apr2016_V4 (10nM) | 0.2 | 20 |
2X KAPA SYBG MM | 50 | 5000 |
AP1V4U (100uM) | 0.4 | 40 |
AP2V4 (100uM) | 0.4 | 40 |
H2O | 49 | 4900 |
Total | 100 | 10,000 |
Program 98C 1min -> (98C 30sec -> 55C 45sec -> 72C 45sec) x 14 -> 72C 2min -> 4C hold
- Did in regular thermocyclers
EtOH Precipitation
- 8 15-ml tubes (with 12 wells (1.5 strips) of PCR product each) for
- 1,200ul PCR product
- 3000ul 100% EtOH
- 4ul GlycoBlue
- 120ul 3M NaOAc pH 5.2-5.5
- Vortexed and put in -80C for 30 min
- 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 8 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
- 8 columns elute 50ul each
- Nanodrop: 313.6 ng/uL x 400uL = 125.44ug
Lambda Exo
- Divide into 8 pcr tubes of 100ul (ideally should be <10ug each)
Components | Volume |
Amplicon | 50 |
10X Lambda Exo Buffer | 10 |
Lambda Exonuclease | 10 |
H2O | 30 |
Total | 100 |
- Incubated at 37C for 2hr (normally 1hr)
- Purified with 8 Zymo ssDNA/RNA columns
- Dropped one column so only 7
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 118.7ng/ul x 280ul = 33.24ug
Remove Amplification Adapters
USER
- Split into 5 PCR tubes and add 5ul USER
Components | Volume |
SS-amplicon | 56 |
USER | 5 |
10X DpnII Buffer | 8 |
H2O | 11 |
Total | 80 |
- Incubate at 37C for 2.5 hours
DpnII
- Added 15ul of the following
Components | Volume |
10X DpnII Buffer | 2 |
100uM RE-DpnII_V4 guide oligo | 5 |
H2O | 8 |
- Incubated at 94C for 2 min, then 37C for 3 min
- Added 5ul DpnII
- Incubated at 37C for overnight (~15hrs)
Zymo Column Purification
- Eluted 20ul each column (100ul total)
- Nanodrop
- 193.7 ng/ul x 100ul = 19.37 ug (% yield)
PAGE Size Selection
- Run 4 gels
- 200V for 40min
Components | 4X Volume |
V4 Probes | 95 |
TBE-Urea Buffer 2X | 95 |
Components | 4X Volume |
Low Mass Ladder | 4 |
TBE-Urea Buffer 2X | 20 |
H2O | 16 |
Total | 40 |
File:2016-06-02 TB12kApr2016 V4 SizeSelect Gel1.jpg
File:2016-06-02 TB12kApr2016 V4 SizeSelect Gel2.jpg
File:2016-06-02 TB12kApr2016 V4 SizeSelect Gel3.jpg
File:2016-06-02 TB12kApr2016 V4 SizeSelect Gel4.jpg
EtOH Precipitation
- Put cut out gel in 8 0.5mL tubes (with holes at bottom) and centrifuged the tube at 15,000 rpm, for 3 min at RT.
- Transferred the gel remaining in 0.5 mL tube to 1.5 mL tube below with pipette tip.
- Added 450 ul of 1X TE buffer to each
- Vortexed for 60min at 37 C in incubator
- Centrifuged at 15,000 rpm for 3 min at RT
- Transferred the clear spnt. to 2 Nanosep columns and centrifuged at 15,000 rpm for 3 min
- Transferred the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min
- Transferred spnt to fresh 1.5 mL tube (~3500ul)
- Precipitated in 8 1.5ml tubes with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 45ul of 3M NaoAc pH 5.2
- Vortexed and placed the 8 tubes at -80C for 30min
- Spun 8 tubes at 10,000rpm at 4C for 30min
- Discard supernatant and add 750ul cold 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 combine
Qubit ssDNA
20.2ng/ul x 74ul = 1,494.8ng 20.2ng/ul / (157nt x 303.7Da/nt + 79Da) = 423nM-->