Daniel:Notebook/ComboLock/2017-6-16: Difference between revisions
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>Djacobse |
>Djacobse |
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<li>Heat kill with 65C for 20 min</li> | <li>Heat kill with 65C for 20 min</li> | ||
<li>Hold at 12C</ol> | <li>Hold at 12C</ol> | ||
<li>qPCR</li> | <li>qPCR</li> | ||
<ol type="A"> | <ol type="A"> | ||
Line 61: | Line 60: | ||
<li>Add 1 uL AmpR and 2 uL sample according to plate layout</li> | <li>Add 1 uL AmpR and 2 uL sample according to plate layout</li> | ||
<li>Use the following protocol</li> | <li>Use the following protocol</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 together 120 uL TBE and 30 uL 6X dye</li> | |||
<li>Aliquot 10 uL/lane onto parafilm</li> | |||
<li>Add 2 uL correct sample or (1.5 uL for ladder) to aliquots</li> | |||
<li>Add 10 uL aliquots to correct lanes</li> | |||
<li>Run gel at 235V for 24 minutes</li> | |||
<li>Remove gel from case and incubate in TBE buffer with 2 uL SYBR Gold for 3 minutes</li> | |||
<li>Image in gel doc</li> | |||
</ol> | </ol> | ||
</ol> | </ol> |
Revision as of 18:54, 16 June 2017
Antibody Detection Test-Positive Control (Started Wednesday)
Protocol-Part 5 (From Yesterday)
- Bglii digestion
- Make the following reactions; Do not add Bglii yet
- Aliquot 12 uL master mix into new tube
- Add 7 uL appropriate sample
- Mix with vortexer, spin down, and add 1 uL Bglii
- Incubate 2 hr 37C
- Heat kill with 65C for 20 min
- Hold at 12C
- qPCR
- Make the following 7.2X master mix (1X)
- 7.2 uL 10 uM AmpF6.4Sol
- 151.2 uL (21) nfH2O
- 180 uL 2X Kapa SYBR Fast Master Mix
- Add 47 uL master mix to each lane
- Add 1 uL AmpR and 2 uL sample according to plate layout
- Use the following protocol
- 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 together 120 uL TBE and 30 uL 6X dye
- Aliquot 10 uL/lane onto parafilm
- Add 2 uL correct sample or (1.5 uL for ladder) to aliquots
- Add 10 uL aliquots to correct lanes
- Run gel at 235V for 24 minutes
- Remove gel from case and incubate in TBE buffer with 2 uL SYBR Gold for 3 minutes
- Image in gel doc
Reagent | Single Rxn Vol | Master Mix (6.2X) |
RCA Rxn | 7 | 0 |
10X Buffer 3.1 | 2 | 12.4 |
Bglii | 1 | 6.2 |
nfH2O | 10 | 62 |
Total | 20 | 80.6 |