Daniel:Notebook/ComboLock/2016-7-26
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Latch 100 and Dextran Test (Started yesterday)[edit]
Protocol[edit]
- Circularization
- Heat inactivate enzyme by incubating for 2 minutes at 94C
- Exonuclease Digestion
- Prepare exonuclease I/III mix by mixing 10 uL exonuclease I (20 units/μL) and 2 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
- qPCR
- Set up reaction according to table
- Master Mix
- 177.5 uL KAPA SyberFast MM
- 124.25 uL nfH2O
- 3.55 uL AmpF 6.4 100 uM
- Add 43 uL Master Mix to each well
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x20
- 72C 2 min
- 16C hold
- 2016-07-26-Latch100X-DextranqPCR.png
qPCR Curve
- 2016-07-26-Latch100X-Dextran.png
Gel Image
- Size Selection
- Combine 20 uL sample from lanes 1-5
- Add 20 uL 6X loading dye
- Aliquot 30 uL each into 4 lanes
- 2 uL ladder with 5 uL gel loading dye and 23 uL TBE buffer
- Run gel for 25 min at 250V
- Stain with 3 uL SYBR Gold for 3 min
- Image in gel doc; cut out selected bands and place in 0.5 mL tube with hole in the bottom inside a 1.5 mL tube
- 2016-07-26-Latch100X-Dextran-SizeSelect.png
Before Image
- 2016-07-26-Latch100X-Dextran-SizeSelect-After.png
After Image
- Centrifuge gel at 14000rpm for 1.5 minutes
- Remove 0.5 mL tube and add 500 uL TE buffer to shredded gel
- Incubate at 37C for at least an hour with vigorous shaking
- Centrifuge at 14000rpm for 1.5 minutes
- Transfer supernatant to a nanosep column
- Centrifuge at 14000rpm for 1.5 minutes
- Transfer flow through to a 2 mL tube
- Ethanol Precipitation
- Add 1.25 mL 100% EtOH, 50 uL 3M NaOAc, and 2 uL glycoblue to sample
- Incubate overnight at -80C; continued tomorrow
Sample | Lane | AmpR Index | Cells | 2X Kapa SYBR qPCR MM | 100 uM AmpF 6.4 | 10 uM AmpR6.3-IndXX | H2O | Total Volume (uL) |
Sample 1 (Hs-Control) | A1 | 9 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 2 (Hs-Dextran) | B1 | 10 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 3 (Hs-Latch 100X) | C1 | 11 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 4 (Hs-Neg. Control) | D1 | 12 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 5 (mm-Control) | E1 | 13 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 6 (NTC) | F1 | 14 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 7 (No sample) | G1 | 15 | 0 | 25 | 0.5 | 5 | 19.5 | 50 |