Daniel:Notebook/ComboLock/2017-1-19: Difference between revisions
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<gallery perrow=2 heights=300px widths=300px> | <gallery perrow=2 heights=300px widths=300px> | ||
File:|qPCR Curve | File:20170119-qPCR-v3Oligos-AmpFAmpR.png|qPCR Curve-AmpF and AmpR | ||
File:|qPCR Curve | File:20170119-qPCR-v3Oligos-Primer12RCAmpR.png|qPCR Curve-Primer12RC and AmpR | ||
File:2017-01-19-v3Oligos-RCA-2.png|Gel Image-AmpF and AmpR | File:2017-01-19-v3Oligos-RCA-2.png|Gel Image-AmpF and AmpR | ||
File:2017-01-19-v3Oligos-NoAmpF.png|Gel Image-Primer12RC and AmpR | File:2017-01-19-v3Oligos-NoAmpF.png|Gel Image-Primer12RC and AmpR | ||
</gallery> | </gallery> |
Revision as of 21:02, 19 January 2017
Version 3 Oligos (Started Thursday, Jan 13)
RCA Repeat (Started Yesterday)
- qPCR
- Make qPCR master mix according to following table
- Add 47 (MM1) or 48 (MM2) uL master mix to each well
- Add 2 uL sample and 1 uL appropriate Reverse Primer (10 uM, MM1 only) according to table
- 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 80 uL TBE, 20 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
Master Mix | Primer Pair | Forward Primer (10 uM) uL | Reverse Primer (10 uM) uL | uL nfH2O | uL Kapa SYBR Fast Master Mix 2X |
MM1 | AmpF/AmpR | 9.2 | NA | 193.2 | 230 |
MM2 | Primer12RC/AmpRInd25 | 9.2 | 9.2 | 193.2 | 230 |
Sample | Condition | Lane | Primer Pair | AmpR Index | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 10 uM Forward Primer | 10 uM Reverse Primer | H2O | Total Volume (uL) | Master Mix |
Sample 1A | Sample | A1 | AmpF/AmpR | 21 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 1B | Sample | A2 | AmpF/AmpR | 21 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 2A | No Template | A3 | AmpF/AmpR | 22 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 2B | No Template | A4 | AmpF/AmpR | 22 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 3A | No C Probe | A5 | AmpF/AmpR | 23 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 3B | No C Probe | A6 | AmpF/AmpR | 23 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 4A | No PCCB Phosphate | A7 | AmpF/AmpR | 24 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 4B | No PCCB Phosphate | A8 | AmpF/AmpR | 24 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 1A | Sample-PostRCA | H1 | Primer12RC/AmpR | 25 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 1B | Sample-PostRCA | H2 | Primer12RC/AmpR | 25 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 4A | No PCCB Phosphate | H3 | Primer12RC/AmpR | 25 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 4B | No PCCB Phosphate | H4 | Primer12RC/AmpR | 25 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 1A | Sample-PreRCA | H5 | Primer12RC/AmpR | 25 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 1B | Sample-PreRCA | H6 | Primer12RC/AmpR | 25 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 4A | No PCCB PO4-PreRCA | H7 | Primer12RC/AmpR | 25 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 4B | No PCCB PO4-PreRCA | H8 | Primer12RC/AmpR | 25 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
NTC | qPCR Primers Only | D1 | AmpF/AmpR | 26 | 2 | 25 | 1 | 1 | 23 | 50 | MM1 |
NTC | qPCR Primers Only | D2 | Primer12RC/AmpR | 25 | 2 | 25 | 1 | 1 | 23 | 50 | MM2 |
Results
- 20170119-qPCR-v3Oligos-AmpFAmpR.png
qPCR Curve-AmpF and AmpR
- 20170119-qPCR-v3Oligos-Primer12RCAmpR.png
qPCR Curve-Primer12RC and AmpR
- 2017-01-19-v3Oligos-RCA-2.png
Gel Image-AmpF and AmpR
- 2017-01-19-v3Oligos-NoAmpF.png
Gel Image-Primer12RC and AmpR