Daniel:Notebook/ComboLock/2017-3-23: Difference between revisions
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Use the AmpF/AmpR gel as a reference point in the future. Also, positive controls for qPCR and the like should be done at a 1:10 dilution of the original. The original concentration, via Qubit is: | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#80FF00;font-size:12pt;font-weight:bold" align="center" | |||
| width="85" height="30" | Qubit | |||
| width="85" | Dil | |||
| width="200" | Meas | |||
| width="105" | Amt | |||
| width="105" | Average (ng/uL) | |||
| width="105" | uM | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | x1 | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 118 | |||
| align="center" align="center" valign="bottom" | 118 | |||
| align="center" align="center" valign="bottom" | | |||
| align="center" align="center" valign="bottom" | | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | x2 | |||
| align="center" align="center" valign="bottom" | 5 | |||
| align="center" align="center" valign="bottom" | 18.3 | |||
| align="center" align="center" valign="bottom" | 91.5 | |||
| align="center" align="center" valign="bottom" | | |||
| align="center" align="center" valign="bottom" | | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | x3 | |||
| align="center" align="center" valign="bottom" | 10 | |||
| align="center" align="center" valign="bottom" | 8.94 | |||
| align="center" align="center" valign="bottom" | 89.4 | |||
| align="center" align="center" valign="bottom" | 99.6 | |||
| align="center" align="center" valign="bottom" | 1.61 | |||
|} | |||
[[Category:ComboLock]] [[Category:20170322]] | [[Category:ComboLock]] [[Category:20170322]] |
Revision as of 22:08, 23 March 2017
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
Circular Product target (104bp)
- 2017-03-23-CPProd-qPCR-AmpF-AmpR.png
AmpF/AmpR (221 bp)
Use the AmpF/AmpR gel as a reference point in the future. Also, positive controls for qPCR and the like should be done at a 1:10 dilution of the original. The original concentration, via Qubit is:
Qubit | Dil | Meas | Amt | Average (ng/uL) | uM |
x1 | 1 | 118 | 118 | ||
x2 | 5 | 18.3 | 91.5 | ||
x3 | 10 | 8.94 | 89.4 | 99.6 | 1.61 |