Daniel:Notebook/ComboLock/2016-11-1
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Circularization Optimization Testing (Started Yesterday)[edit]
Sample Matrix[edit]
Sample | Polymerase | Incubation Time | RCA Y/N |
Sample 1 (AB) | Klentaq | ON | Y |
Sample 2 (AB) | Klentaq | ON | N |
Sample 3 (AB) | Klentaq | 2 hr | Y |
Sample 4 (AB) | Klentaq | 2 hr | N |
Sample 5 (AB) | Phusion | ON | Y |
Sample 6 (AB) | Phusion | ON | N |
Sample 7 (AB) | Phusion | 2 hr | Y |
Sample 8 (AB) | Phusion | 2 hr | N |
Protocol[edit]
- Circularization (all samples)
- Heat inactivate enzyme by incubating for 2 minutes at 94C
- Exonuclease Digestion
- Prepare exonuclease I/III mix by mixing 30 uL exonuclease I (20 units/μL) and 6 uL exonuclease III (100 units/μL) in 1:1 ratio
- Add 2 μL of exonuclease I/III mix to reaction
- Mix the reaction by swirling pipette around the well 5 times
- Incubate reaction at 37 ºC for 2 hours
- Heat inactivate enzyme by incubating at 94C for 5 minutes
- Rolling Circle Amplification (Samples 1,3,5,7; Hold samples 2,4,6,8 at 4C)
- Prepare 8.1X Master mix according to table below
- Add sample to new reaction tubes according to following table
- Incubate at 37C for 3 hours
- Incubate at 65C for 10 minutes
- Hold at 10C
- qPCR
- Set up reaction according to table below
- Make master mixes according to following recipes
- 535.5 uL nfH2O
- 637.5 uL SYBR Fast
- 25.5 uL 10 uM AmpF
- Add 47 uL master mix to each well
- 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
Reagent | uL Added | Master Mix (8.1X) |
Template | 5 | NA |
RCA Primer (100 uM) | 2.5 | 20.25 |
dNTP (1 mM) | 0.8 | 6.48 |
10X Buffer | 2 | 16.2 |
Phi29 | 1 | 8.1 |
nfH2O | 8.7 | 70.47 |
Total | 20 | 121.5 |
qPCR Sample Table[edit]
Sample | Condition | RCA? | 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 | K-ON-Y | N | A1 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1B | K-ON-Y | N | A2 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2A | K-ON-N | N | A3 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2B | K-ON-N | N | A4 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3A | K-2H-Y | N | A5 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3B | K-2H-Y | N | A6 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4A | K-2H-N | N | A7 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4B | K-2H-N | N | A8 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 5A | P-ON-Y | N | F1 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 5B | P-ON-Y | N | F2 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 6A | P-ON-N | N | F3 | 25 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 6B | P-ON-N | N | F4 | 25 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 7A | P-2H-Y | N | F5 | 26 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 7B | P-2H-Y | N | F6 | 26 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 8A | P-2H-N | N | F7 | 27 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 8B | P-2H-N | N | F8 | 27 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1A | K-ON-Y | Y | H1 | 28 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1B | K-ON-Y | Y | H2 | 28 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3A | K-2H-Y | Y | H3 | 29 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3B | K-2H-Y | Y | H4 | 29 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 5A | P-ON-Y | Y | H5 | 30 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 5B | P-ON-Y | Y | H6 | 30 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 7A | P-2H-Y | Y | H7 | 31 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 7B | P-2H-Y | Y | H8 | 31 | 2 | 25 | 1 | 1 | 21 | 50 |
NTC | NA | N | C1 | 33 | 2 | 25 | 1 | 1 | 21 | 50 |
Results[edit]
- 20161101-qPCR-Circularization-KvsP.png
Klentaq (Blue) vs Phusion (Red)
- 20161101-qPCR-Circularization-IncTime.png
Incubation Time; Overnight (Blue) vs 2 Hour (Red)
- 20161101-qPCR-Circularization-RCACompare.png
Rolling Circle Amplification (3hr); Before (Red) and After (Blue)
- 2016-11-01-CircularizationTest-Klentaq.png
Gel image Post-Klentaq
- 2016-11-01-CircularizationTest-Phusion.png
Gel image Post-Phusion
- 2016-11-01-CircularizationTest-PostRCA.png
Gel image Post-RCA
- 2016-11-01-CircularizationTest-PostRCA-Edit.png
Gel image Post-RCA; Lines edit