Matt:LabNotes/2017-6-6: Difference between revisions
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>Mzcai (Created page with "==Agi15k_Feb2017_V4 Probe Production== *Production of Padlock Probes (V4) Matt:LabNotes/2017-2-8 ===Production PCR=== *Matt:LabNotes/2017-3-30 | 25nM 1st round amplicon...") |
>Mzcai |
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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: | ~400ul of V4 probes: 248.3 ng/ul => ~99.3 ug | ||
===Lambda Exonuclease Digestion=== | ===Lambda Exonuclease Digestion=== | ||
Line 63: | Line 63: | ||
*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:<!-- | ||
**2hr incubation: 81.7 ng/ul x 160ul = 13 ug | **2hr incubation: 81.7 ng/ul x 160ul = 13 ug | ||
Revision as of 19:50, 7 June 2017
Agi15k_Feb2017_V4 Probe Production
- Production of Padlock Probes (V4) Matt:LabNotes/2017-2-8
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 3,000rpm 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 5 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:
~400ul of V4 probes: 248.3 ng/ul => ~99.3 ug
Lambda Exonuclease Digestion
- Divide into 4 pcr tubes of ~100ul with total amplicon of <10ug each
Components | Volume |
Amplicon | 96 |
10X Lambda Exo Buffer | 12 |
Lambda Exonuclease | 12 |
Total | 120 |
- Incubate 4 tubes at 37C for 2hr
- Purified with 8 Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop: