Daniel:Notebook/ComboLock/2016-10-18
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Cell Trial Padlock0201 (Started Yesterday)
Protocol
- Circularization
- Heat inactivate enzyme by incubating for 2 minutes at 94C
- Prepare exonuclease I/III mix by mixing 7.5 uL exonuclease I (20 units/μL) and 1.5 uL exonuclease III (100 units/μL) in 1:1 ratio
- Add 2 μL of exonuclease I/III mix to reaction
- Mix the reaction by swirling pipette around the well 5 times
- Incubate reaction at 37 ºC for 2 hours
- Heat inactivate enzyme by incubating at 94C for 5 minutes
- Prepare the following master mix
- 89.25 uL nfH2O
- 2.55 uL 100 uM AmpF6.4Sol
- 127.5 uL Kapa SYBR Fast MM
- Add 43 uL master mix to each well
- Add sample and AmpR indexes according to sample table below
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x24
- 72C 2 min
- 16C hold
Sample | Condition | Lane | AmpR Index | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 100 uM AmpF6.4Sol | 10 uM AmpR6.3-IndXX | H2O | Total Volume (uL) |
Sample 1 | Exp. Rep 1 | A1 | 20 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 2 | Exp. Rep 2 | B1 | 20 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 3 | No C Probes | C1 | 21 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 4 | No Cells | D1 | 22 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
NTC | No qPCR sample | E1 | 23 | 0 | 25 | 0.5 | 5 | 19.5 | 50 |
- Mix 56 uL TBE, 14 uL 6x loading dye
- Aliquot 10 uL per sample/ladder lane onto parafilm
- Add 2 uL of sample or ladder to correct drop
- Load 10 uL in to well
- Run gel for 22 minutes at 250V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc