Daniel:Notebook/ComboLock/2017-1-26: Difference between revisions
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File:2017-01-26-RCATest-Epicentre.png|Gel image | File:2017-01-26-RCATest-Epicentre.png|Gel image | ||
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[[Category:ComboLock]] [[Category:20170123]] |
Revision as of 00:55, 27 January 2017
RCA Test (Started Monday)
Epicentre Test (Started Yesterday)
Sample Matrix
- qPCR
- Make 5.2X qPCR master mix according to following recipe
- 109.2 uL nfH2o
- 5.2 uL AmpF
- 130 uL 2X Kapa SYBR Fast Master Mix
- Add 47 uL master mix to each well
- Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) 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 64 uL TBE, 16 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
- Size Select Gel
- Mix 20 uL sample from each tech rep lane (A and B) and 8 uL 6x loading dye into separate tubes for each sample (Samples 1A and 1B)
- Mix 4 uL ladder, 4 uL 6x dye, and 16 uL TBE in ladder tube
- Aliquot 48/24 uL per sample/ladder lane into each 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
- Cut out appropriate bands with scalpel; put in 0.5 mL tube with hole in the bottom inside a 1.5 mL tube
- Image in gel doc
- 2017-01-26-RCATest-Epicentre-SizeSelect.png
Before Image
- 2017-01-26-RCATest-Epicentre-SizeSelect-After.png
After Image
- Centrifuge at 12000 rpm for 1.5 minutes
- Add 500 uL TE to each sample
- Incubate 4 hours at 37C with vigorous shaking;
- Centrifuge tubes at 12000 rpm for 1.5 minutes to bring gel to bottom
- Extract supernatant and add to nanosep column
- Centrifuge at 12000 rpm for 1.5 minutes; collect supernatant
- Ethanol Precipitation
- Add 1250 uL 100% EtOH, 50 uL 3M NaOAc, and 1.5 uL glycoblue to each sample
- Incubate at -80C overnight; continued tomorrow
- 20170126-qPCR-RCATest-Epicentre.png
qPCR curve
- 2017-01-26-RCATest-Epicentre.png
Gel image
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 6AC | Epicentre | A1 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 6BC | Epicentre | A2 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 6AD | Epicentre 1:10 | A3 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 6BD | Epicentre 1:10 | A4 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
NTC | A5 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
Library Prep
Those bands are interesting to me. Time to sequence.