Matt:LabNotes/2016-6-7: 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 2X V4 master mix {| {{table}} | align="cen...") |
>Mzcai |
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**120ul 3M NaOAc pH 5.2-5.5 | **120ul 3M NaOAc pH 5.2-5.5 | ||
*Vortexed and put in -80C for overnight | *Vortexed and put in -80C for overnight | ||
*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 | ||
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*Recombined and measured ssDNA with Nanodrop: | *Recombined and measured ssDNA with Nanodrop: | ||
**91.1ng/ul x 320ul = 29.15ug (50.7% yield) | **91.1ng/ul x 320ul = 29.15ug (50.7% yield) | ||
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===Remove Amplification Adapters=== | ===Remove Amplification Adapters=== | ||
====USER==== | ====USER==== |
Revision as of 20:51, 9 June 2016
TB12k_Apr2016 V4 Probe Preparation
Production PCR
- Make 2X V4 master mix
Components | Volume (1X) | Volume (50X) |
First round amplicon TB12k_Apr2016_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 |
Program 98C 1min -> (98C 30sec -> 55C 45sec -> 72C 45sec) x 14 -> 72C 2min -> 4C hold
- No curve because 100ul reaction volume only works in Biorad PCR machines
EtOH Precipitation
- 8 15-ml tubes (with 24 wells (3 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 overnight
- 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: 287.3 ng/uL x 400uL = 114.9ug
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
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 91.1ng/ul x 320ul = 29.15ug (50.7% yield)