Matt:LabNotes/2015-3-25
Jump to navigation
Jump to search
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 Elution buffer
- 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
Repeat Production PCR
- 10nM 1st round amplicons
- V4 master mix made in 1 15ml centrifugal tube
- Poured into trough/reservoir and used multi-channel pipette to save time pipetting into pcr 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
- Tried using reservoir & multi-channel pipette removing product from 6 strips of pcr tubes but spilled ~1ml (product+binding buffer) pouring from reservoir into 50ml tubes
- 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 400ul H2O
- Somehow final volume is greater than 400ul
- Measured concentration with Nanodrop:
~410ul of V4 probes: 76.1 ng/ul => 31.2ug
Lambda Exonuclease Digestion
- Divide into 9 pcr tubes of ~110ul with total amplicon of <10ug each
Components | Volume |
Amplicon | 96 |
10X Lambda Exo Buffer | 12 |
Lambda Exonuclease | 12 |
Total | 120 |
- Incubated at 37C for 1hr
- Purified with 9 Zymo ssDNA/RNA columns
- Eluted with 30ul each
- Recombined and measured ssDNA with Nanodrop:
- ng/ul x 270ul = ug (% yield)