Daniel:Notebook/ComboLock/2017-3-21
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Circularization Comparison (T4 Poly/Ligase; Started Wednesday)[edit]
Bgl Digestion[edit]
Because I CAN. Also, will determine if there is an improvement in signal post-digestion.
Sample Matrix
Sample Origin | Condition | Sample Numbers |
F | T4 Ligase | 1,4 |
G | Phusion/Amp (Unpurified) | 1,4 |
H | Phusion/AmpHS (Unpurified) | 1,4 |
- Bglii Digestion
- Use samples 1 (Experiment) and 4 (No phosphate) for Bglii digest
- Make the following 4.2X master mix
- Aliquot 14 uL master mix into new tubes
- Add 5 uL appropriate sample to each tube
- Use the following thermocycler program
- 5 min 95C
- 10 min 50C
- 1 hr 37C; When this cycle starts add 1 uL Bglii
- Heat kill with 65C for 20 min
- qPCR
- Make qPCR master mix according to following recipe
- 319.2 uL nfH2O
- 15.2 uL AmpF
- 15.2 uL AmpR-Ind22
- 380 uL Kapa SYBR Fast 2X Master Mix
- Add 48 uL master mix to each well
- Add 2 uL sample according to plate layout File:PlateLayout-T4Circ-BgliiDigest-20170321.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
- 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 | Single Rxn Vol | 14.2X MM uL |
RCA Reaction Product | 5 | 0 |
Bglii Cutter Primer (10 uM) | 5 | 71 |
10X Buffer 3.1 | 2 | 28.4 |
Bglii | 1 | 0 |
nfH2O | 7 | 99.4 |
Total | 20 | 198.8 |
Results[edit]
- 20170321-qPCR-T4Circ-PostBglii.png
qPCR curves
- 2017-03-21-T4Circ-PostBglii-T4Samples.png
Gel image-T4 Ligase samples (F)
- 2017-03-21-T4Circ-PostBglii-APSamples.png
Gel image-Amp/Phusion samples (G,H)