Daniel:Notebook/ComboLock/2017-3-9: Difference between revisions
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[[Image:PlateLayout-20170309-BoltTest.png|600px]] | [[Image:PlateLayout-20170309-BoltTest.png|600px]] | ||
<li>qPCR Cycles</li> | |||
<ol type="a"> | |||
<li>95C 3 min</li> | |||
<li>95C 3 sec</li> | |||
<li>55C 30 sec</li> | |||
<li>72C 20 sec</li> | |||
<li>plate read</li> | |||
<li>goto b x30</li> | |||
<li>72C 2 min</li> | |||
<li>16C hold</li> | |||
</ol></ol> | |||
<li>TBE Gel</li> | |||
<ol type="A"> | |||
<li>Mix 96 uL TBE, 24 uL 6x loading dye</li> | |||
<li>Aliquot 10 uL per sample/ladder lane onto parafilm</li> | |||
<li>Add 2 uL of sample or ladder to correct drop</li> | |||
<li>Load 10 uL in to well</li> | |||
<li>Run gel for 23 minutes at 230V</li> | |||
<li>Open gel and stain with 3 uL SYBR Gold for 3 minutes</li> | |||
<li>Rinse gel and image in gel doc</li> | |||
</ol></ol> | |||
[[Category:ComboLock]] [[Category:20170308]] | [[Category:ComboLock]] [[Category:20170308]] |
Revision as of 01:00, 9 March 2017
Bolt Test (Started Yesterday)
Protocol
- Bolt/Latch Hybridization
- Wash twice with 100 uL wash buffer
- Elution
- Add 50 uL wash buffer to each sample
- Cook at 95C for 15 minutes
- qPCR
- Make master mixes according to following recipes
- Add 48 uL master mix to each well
- Add 2 uL sample according to plate layout File:PlateLayout-20170309-BoltTest.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 96 uL TBE, 24 uL 6x loading dye
- Aliquot 10 uL per sample/ladder lane onto parafilm
- Add 2 uL of sample or ladder to correct drop
- Load 10 uL in to well
- Run gel for 23 minutes at 230V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
Single Rxn | MMC1 | MMC2 | MMP | |
Primer Pair | NA | p24/p4RC | p6/p12RC | p24/p12RC |
uL Fwd Primer (10 uM) | 1 | 10.2 | 10.2 | 10.2 |
uL Rev Primer (10 uM) | 1 | 10.2 | 10.2 | 10.2 |
uL nfH2O | 21 | 214.2 | 214.2 | 214.2 |
uL Kapa SYBR Fast | 25 | 255 | 255 | 255 |
Total | 48 | 489.6 | 489.6 | 489.6 |