Daniel:Notebook/ComboLock/2017-3-7: Difference between revisions
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[[Image:PlateLayout-20170307-Padlock0601-MaxRCA.png|600px]] | [[Image:PlateLayout-20170307-Padlock0601-MaxRCA.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> | |||
==Protocol-Part 3-Post Bglii== | |||
<li>qPCR</li> | |||
<ol type="A"> | |||
<li>Make 9.2X qPCR master mix according to following recipe</li> | |||
<ol type="a"> | |||
<li>193.2 uL nfH2O</li> | |||
<li>230 uL SYBR Fast</li> | |||
<li>9.2 uL 10 uM AmpF</li> | |||
<li>9.2 uL 10 uM AmpR-Ind21</li> | |||
</ol> | |||
<li>Add 48 uL master mix to each well</li> | |||
<li>Add 2 uL sample according to plate layout</li> | |||
[[Image:|600px]] | |||
<li>qPCR Cycles</li> | <li>qPCR Cycles</li> | ||
Line 83: | Line 112: | ||
<li>16C hold</li> | <li>16C hold</li> | ||
</ol></ol></ol> | </ol></ol></ol> | ||
===Results=== | ===Results=== |
Revision as of 19:41, 7 March 2017
Padlock0601 Test (Started Wed March 1)
Protocol-Part 2 (Protocol Started Yesterday)
- 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 16.2X qPCR master mix according to following recipe
- 340.2 uL nfH2O
- 405 uL SYBR Fast
- 16.2 uL 10 uM AmpF
- 16.2 uL 10 uM AmpR-Ind20
- Add 48 uL master mix to each well
- Add 2 uL sample according to plate layout File:PlateLayout-20170307-Padlock0601-MaxRCA.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
- qPCR
- Make 9.2X qPCR master mix according to following recipe
- 193.2 uL nfH2O
- 230 uL SYBR Fast
- 9.2 uL 10 uM AmpF
- 9.2 uL 10 uM AmpR-Ind21
- Add 48 uL master mix to each well
- Add 2 uL sample according to plate layout [[Image:|600px]]
- 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 Vol | 8.2X MM uL |
RCA Reaction Product | 5 | 0 |
Bglii Cutter Primer (10 uM) | 0 | 0 |
10X Buffer 3.1 | 2 | 16.4 |
Bglii | 1 | 8.2 |
nfH2O | 12 | 98.4 |
Total | 20 | 114.8 |
Protocol-Part 3-Post Bglii
Results
- 2017-03-07-Padlock0601-MaxRCA-Unpurified.png
Gel image-Unpurified RCA input
- 2017-03-07-Padlock0601-MaxRCA-Purified.png
Gel image-Purified RCA input