Matt:LabNotes/2015-2-2: Difference between revisions
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>Mzcai m (→Production PCR) |
>Mzcai mNo edit summary |
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'''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold | '''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold | ||
[[File: | 800px]] | [[File: | 800px]] | ||
===EtOH Precipitation=== | ===EtOH Precipitation=== | ||
*12 5-ml tubes (with 8 wells of PCR product each) for V4 | *12 5-ml tubes (with 8 wells of PCR product each) for V4 | ||
Line 40: | Line 40: | ||
*Discarded supernatant and air-dried for 5 min in hood | *Discarded supernatant and air-dried for 5 min in hood | ||
*Resuspended DNA with 100ul H2O | *Resuspended DNA with 100ul H2O | ||
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===Qia Column Purification=== | ===Qia Column Purification=== | ||
*Purified | *Purified 12 tubes of 100ul in 12 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: | ||
~ | ~580ul of V4 probes: 93.2 ng/ul => ~27 ug | ||
===Lambda Exonuclease Digestion=== | ===Lambda Exonuclease Digestion=== |
Revision as of 00:44, 3 February 2015
CA12k_Nov2014_V4 Probe Production
- Production of Padlock Probes (V4) Matt:LabNotes/2014-11-19#Assembly_of_oligo_pool_for_ordering
- Do double of what I normally do (96 100ul PCR reactions instead of 48)
Production PCR
- 10nM 1st round amplicons
- V4 master mix made in 2 5ml tubes
Components | Volume (1X) | Volume (100X) |
First round amplicon CA12k_Nov14_V7 (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 | 10000 |
Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold [[File: | 800px]]
EtOH Precipitation
- 12 5-ml tubes (with 8 wells of PCR product each) for V4
- 800ul PCR product
- 2000ul 100% EtOH
- 2.7ul GlycoBlue
- 80ul 3M NaOAc pH 5.2-5.5
- Vortexed and put in -80C for 30min
- 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 6 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