Daniel:Notebook/ComboLock/2017-3-23
Jump to navigation
Jump to search
Circular Product Production (Started Yesterday)
Protocol
- qPCR
- Make qPCR master mix according to following recipes
- Add 48 uL master mix to each well
- Add 2 uL sample according to plate layout File:PlateLayout-20170323-CPProduction.png
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x30
- 72C 2 min
- 16C hold
- Qubit Measurement
- Add 995 uL buffer and 5 uL ssdye to tube
- Add 190 uL buffer and 10 uL correct standard to standard tubes
- Add 199 uL buffer and 1 uL sample to sample tubes
- Let sit 5-10 minutes in the dark
- Measure in Qubit
- TBE Gel
- Mix 120 uL TBE and 40 uL 6X dye
- Aliquot 10 uL per sample on to parafilm
- Add 2 uL sample or 1.5 uL ladder to aliquots
- Mix and add 10 uL per lane to gel
- Run gel at 230V for 24 minutes
- Add 2 uL SYBR gold and incubate on shaker for 3 minutes
- Rinse and image in gel doc
Reagent | 1X Rxn | MM-Amp (6.2X) | MM-Prod (7.2X) |
Primer Pair | NA | AmpF/AmpR | p24/p12RC |
Forward Primer (10 uM) | 1 | 6.2 | 7.2 |
Forward Primer (10 uM) | 1 | 6.2 | 7.2 |
Kapa SYBR Fast Master Mix (2X) | 25 | 155 | 180 |
nfH2O | 21 | 130.2 | 151.2 |
Total | 48 | 297.6 | 345.6 |
Results
- 20170323-qPCR-CPProd.png
qPCR curves
- 2017-03-23-CPProd-qPCR-CPTarget.png
Gel image-T4 Ligase samples (F)
- 2017-03-23-CPProd-qPCR-AmpF-AmpR.png
Gel image-Amp/Phusion samples (G,H)