Matt:LabNotes/2017-5-20: Difference between revisions
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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 10,000rpm at 4C for 30 min | *Centrifuged at 10,000rpm 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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*Purified 8 tubes of 100ul in 8 columns following Qiagen protocol | *Purified 8 tubes of 100ul in 8 columns following Qiagen protocol | ||
*Eluted each column with 50ul and combined all into one 1.5ml | *Eluted each column with 50ul and combined all into one 1.5ml | ||
*Measured concentration with Nanodrop: | *Measured concentration with Nanodrop:<!-- | ||
~400ul of V4 probes: 327.7 ng/ul => ~131 ug | ~400ul of V4 probes: 327.7 ng/ul => ~131 ug | ||
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* | *Incubate 2 tubes at 37C for 2hr and 2 tubes at 37C for 1hr | ||
*Purified with 8 Zymo ssDNA/RNA columns | *Purified with 8 Zymo ssDNA/RNA columns | ||
*Eluted with 40ul each | *Eluted with 40ul each | ||
*Recombined and measured ssDNA with Nanodrop: | *Recombined and measured ssDNA with Nanodrop: | ||
**95.1 ng/ul x 320ul = 30.4 ug (46.5% yield) | **1hr incubation: | ||
* | **2hr incubation: 95.1 ng/ul x 320ul = 30.4 ug (46.5% yield) | ||
*Conclusion: | |||
===Remove Amplification Adapters=== | ===Remove Amplification Adapters=== |
Revision as of 22:30, 22 May 2017
Agi15k_Feb2017_V4 Probe Production
- Production of Padlock Probes (V4) Matt:LabNotes/2017-2-8
Notes from last time
- Do 12 or 13 cycles Production PCR
- Experiment with 1hr vs 2hr Lambda digestion
- Think of way to maximize USER and DpnII digestion
Production PCR
- 25nM 1st round amplicons
- V4 master mix made in 2 5ml tubes
Components | Volume (1X) | Volume (100X) |
First round amplicon Agi15k_Feb2017_V4 (25nM) | 0.1 | 10 |
2X KAPA SYBG MM | 50 | 5000 |
AP1V4U (100uM) | 0.4 | 40 |
AP2V4 (100uM) | 0.4 | 40 |
H2O | 49 | 4910 |
Total | 100 | 10000 |
Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 13 -> 72C 2min -> 15C hold
EtOH Precipitation
- 8 5-ml tubes (with 12 wells of PCR product each) for V4
- 1200ul PCR product
- 3000ul 100% EtOH
- 3ul GlycoBlue
- 120ul 3M NaOAc pH 5.2-5.5
- Vortexed and put in -80C for overnight
- Centrifuged at 10,000rpm 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 15 min at 4C
- Discarded supernatant and air-dried for 10 min in hood
- Resuspended DNA with 100ul H2O
Qia Column Purification
- Purified 8 tubes of 100ul in 8 columns following Qiagen protocol
- Eluted each column with 50ul and combined all into one 1.5ml
- Measured concentration with Nanodrop: