Matt:LabNotes/2015-3-25: Difference between revisions
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**Add 50ml DNA Binding Buffer to 10ml product (Use 2 50ml tubes) | **Add 50ml DNA Binding Buffer to 10ml product (Use 2 50ml tubes) | ||
*Elute with 540ul H2O | *Elute with 540ul H2O | ||
**Should elute with < 400ul next time because it won't all fit in column | |||
***Lost about 80ul and ended with low yield (50ug normally after EtOH precipitation and Qia purification) | |||
*Measured concentration with Nanodrop: | *Measured concentration with Nanodrop: | ||
~ | ~460ul of V4 probes: 71.3 ng/ul => 32.8ug | ||
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===Lambda Exonuclease Digestion=== | ===Lambda Exonuclease Digestion=== |
Revision as of 23:06, 25 March 2015
CA12k_Nov2014_V4 Probe Production
- Production of Padlock Probes (V4) Matt:LabNotes/2014-11-19#Assembly_of_oligo_pool_for_ordering
- Do double 96 100ul PCR Reactions
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:032515 CA12kNov14 ProductionPCR V4.JPG
Zymo DNA Clean & Concentrator 100
- Purify 10ml of PCR product with 1 Zymo-spin V Column/Reservoir
- Add 50ml DNA Binding Buffer to 10ml product (Use 2 50ml tubes)
- Elute with 540ul H2O
- Should elute with < 400ul next time because it won't all fit in column
- Lost about 80ul and ended with low yield (50ug normally after EtOH precipitation and Qia purification)
- Should elute with < 400ul next time because it won't all fit in column
- Measured concentration with Nanodrop:
~460ul of V4 probes: 71.3 ng/ul => 32.8ug