Daniel:Notebook/ComboLock/2016-11-4: Difference between revisions
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<li>qPCR</li> | <li>qPCR</li> | ||
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<li>Make master mix according to following recipe</li> | <li>Make master mix according to following recipe</li> | ||
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<li>Add 47 uL master mix to each well</li> | <li>Add 47 uL master mix to each well</li> | ||
<li>Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) according to table in following section</li> | |||
<li>qPCR Cycles</li> | <li>qPCR Cycles</li> | ||
<ol type="a"> | <ol type="a"> |
Revision as of 17:12, 4 November 2016
Antibody Binding Test (Started Yesterday)
Protocol
- qPCR
- Make master mix according to following recipe
- 233.1 uL nfH2O
- 277.5 uL SYBR Fast
- 11.1 uL 10 uM AmpF
- Add 47 uL master mix to each well
- Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) according to table in following section
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x24
- 72C 2 min
- 16C hold
- TBE Gel
- Mix 240 uL TBE, 60 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 22 minutes at 250V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
qPCR Sample Table
Sample | Condition | Lane | AmpR Index | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 10 uM Forward Primer | 10 uM Reverse Primer | H2O | Total Volume (uL) |
Sample 1A | 2 ug BSA | A1 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1B | 2 ug BSA | A2 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2A | No Template | A3 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2B | No Template | A4 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3A | No Antibody | A5 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3B | No Antibody | A6 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4A | 20 ng BSA | A7 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4B | 20 ng BSA | A8 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 5A | 200 pg BSA | H1 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 5B | 200 pg BSA | H2 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
NTC | qPCR NTC | H3 | 25 | 0 | 25 | 1 | 1 | 23 | 50 |