Daniel:Notebook/ComboLock/2017-3-17
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Circularization Comparison (T4 Poly/Ligase; Started Wednesday)[edit]
Protocol-Part 6[edit]
- qPCR
- Make qPCR master mixes according to following recipe
- Add 48 uL master mix to each well
- Add 2 uL sample according to plate layout File:PlateLayout-20170317-T4Circ-PostRCA.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 200 uL TBE and 50 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 | MM1 (23.2X) | MM1-2 (20.2X) | MM+ (C1, 4.2X) |
Primer Pair (fwd/rev) | NA | AmpF/AmpR-Ind21 | AmpF/AmpR-Ind21 | primer24/primer4* |
Forward Primer (10 uM) | 1 | 23.2 | 20.2 | 4.2 |
Reverse Primer (10 uM) | 1 | 23.2 | 20.2 | 4.2 |
nfH2O | 21 | 487.2 | 424.2 | 88.2 |
2X Kapa SYBR Fast | 25 | 580 | 505 | 105 |
Total | 48 | 1113.6 | 969.6 | 201.6 |
Results[edit]
- 20170317-qPCR-T4Circ-T4vsPhu.png
qPCR curves-T4 Ligase protocol vs Amp/Phusion protocol (Post RCA)
- 20170317-qPCR-T4Circ-PurifiedvsUnpurified.png
qPCR curves-Unpurified samples vs Purified Samples (Post RCA)
- 20170317-qPCR-T4Circ-HSvsNormal.png
qPCR curves-Hot start vs Normal (Post RCA)
- 2017-03-17-T4Circ-T4-PostRCA.png
Gel image-T4 Poly/Ligase PostRCA
- 2017-03-20-T4Circ-T4PreRCA-AmpFAmpR.png
Gel image-T4 Poly/Ligase PreRCA (AmpF/AmpR)
- 2017-03-17-T4Circ-Phu-PostRCA.png
Gel image-Phusion/Amp Ligase Hot Start vs Normal PostRCA
- 2017-03-17-T4Circ-PrevsPost.png
Gel image-PreRCA vs PostRCA
- 2017-03-17-T4Circ-PurevsUnpure.png
Gel image-Purified samples vs unpurified (Post PhusionHS)