Daniel:Notebook/ComboLock/2017-3-7
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Padlock0601 Test (Started Wed March 1)[edit]
Protocol-Part 2 (Protocol Started Yesterday)[edit]
- 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
- Mix 160 uL TBE, 40 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 2 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
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[edit]
- 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 File:PlateLayout-20170307-MaxRCA-BgliiDigest.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
- Mix 96 uL TBE, 24 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 2 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
Results[edit]
- 20170307-qPCR-MaxRCA.png
qPCR curves for MaxRCA samples
- 20170307-qPCR-MaxRCA-Adj.png
qPCR curves for MaxRCA samples; adjusted by constant offset to (generally) be non-negative
- 2017-03-07-Padlock0601-MaxRCA-Unpurified.png
Gel image-Unpurified RCA input
- 2017-03-07-Padlock0601-MaxRCA-Purified.png
Gel image-Purified RCA input
- 20170307-qPCR-MaxRCA-Bglii.png
qPCR curves for MaxRCA samples post-Bglii digest
- 2017-03-07-Padlock0601-MaxRCA-Bglii.png
Gel image-Post Bglii Digest