Daniel:Notebook/ComboLock/2017-3-9
Jump to navigation
Jump to search
Bolt Test (Started Yesterday)[edit]
Protocol[edit]
- Bolt/Latch Hybridization
- Wash twice with 100 uL wash buffer
- Elution
- Add 50 uL wash buffer to each sample
- Cook at 95C for 15 minutes
- qPCR
- Make master mixes according to following recipes
- Add 48 uL master mix to each well
- Add 2 uL sample according to plate layout File:PlateLayout-20170309-BoltTest.png
- Note: The parentheses in the descriptions indicate which sample was used
- Note:The colors are according to master mix, targeting either C1 (blue), C2 (red), or product (green)
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x30
- 72C 2 min
- 16C hold
- TBE Gel
- Mix 360 uL TBE, 90 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 23 minutes at 230V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
Single Rxn | MMC1 (11.2X) | MMC2 (11.2X) | MMP (12.2X) | |
Primer Pair | NA | p24/p4RC | p6/p12RC | p24/p12RC |
uL Fwd Primer (10 uM) | 1 | 11.2 | 11.2 | 12.2 |
uL Rev Primer (10 uM) | 1 | 11.2 | 11.2 | 12.2 |
uL nfH2O | 21 | 235.2 | 235.2 | 256.2 |
uL Kapa SYBR Fast | 25 | 280 | 280 | 305 |
Total | 48 | 537.6 | 537.6 | 585.6 |
Results[edit]
- 20170309-qPCR-RFUCurves-BoltTest.png
qPCR curves
- 2017-03-09-BoltTest-C1Target.png
C1 targets
- 2017-03-09-BoltTest-C2Target.png
C2 targets
- 2017-03-09-BoltTest-Product.png
Product targets