Daniel:Notebook/ComboLock/2017-3-17: Difference between revisions
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qPCR
TBE Gel
>Djacobse (Created page with "=Circularization Comparison (T4 Poly/Ligase; Started Yesterday)= Back to Calendar ==Protocol-Part 6== ...") |
>Djacobse |
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| width="200" height="39" | Reagent | | width="200" height="39" | Reagent | ||
| width="85" | Single Rxn | | width="85" | Single Rxn | ||
| width="160" | | | width="160" | MM1 (43.2X) | ||
| width="160" | MM+ (C1, 4.2X) | | width="160" | MM+ (C1, 4.2X) | ||
Revision as of 15:59, 17 March 2017
Circularization Comparison (T4 Poly/Ligase; Started Yesterday)
Protocol-Part 6
- 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
Reagent | Single Rxn | MM1 (43.2X) | MM+ (C1, 4.2X) |
Primer Pair (fwd/rev) | NA | primer12*/AmpR-Ind21 | primer24/primer4* |
Forward Primer (10 uM) | 1 | 15.2 | 4.2 |
Reverse Primer (10 uM) | 1 | 15.2 | 4.2 |
nfH2O | 21 | 319.2 | 88.2 |
2X Kapa SYBR Fast | 25 | 380 | 105 |
Total | 48 | 729.6 | 201.6 |
- 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