Daniel:Notebook/ComboLock/2016-11-8
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Cell Test with RCA (Started Yesterday)[edit]
Protocol[edit]
- Exonuclease Digestion
- Prepare exonuclease I/III mix by mixing 15 uL exonuclease I (20 units/μL) and 3 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
- 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
- Heat kill enzyme by incubating at 65C for 10 minutes
- qPCR
- Make master mix according to following recipe
- 291.1 uL nfH2O
- 227.5 uL SYBR Fast
- 9.1 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
- 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 120 uL TBE, 30 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 A (2.2X) | Master Mix B (2.2X) | Master Mix C (8.2X) |
Template | 5 | 11 | 11 | NA |
RCA Primer (100 uM) | 2.5 | 5.5 | 5.5 | 20.5 |
RCA Primer Name | NA | RCA-CL-v01 | Chris' Primer | RCA-CL-v01 |
dNTP (1 mM) | 0.8 | 1.76 | 1.76 | 6.56 |
10X Buffer | 2 | 4.4 | 4.4 | 16.4 |
Phi29 | 1 | 2.2 | 2.2 | 8.2 |
Phi29 Company | NA | NEB | NEB | Epicentre |
nfH2O | 8.7 | 19.14 | 19.14 | 71.34 |
Total | 20 | 44 | 123 |
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 1AC | Experiment | A1 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1BC | Experiment | A2 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2AC | Experiment | A3 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2BC | Experiment | A4 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3AC | No C Probes | A5 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3BC | No C Probes | A6 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4AC | No Cells | A7 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4BC | No Cells | A8 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1AA | Exp-Epicentre | H1 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1BA | Exp-Epicentre | H2 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2AB | Exp-Primer2 | H3 | 25 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2BB | Exp-Primer2 | H4 | 25 | 2 | 25 | 1 | 1 | 21 | 50 |
NTC | H5 | 26 | 0 | 25 | 1 | 1 | 23 | 50 |
Results[edit]
- 20161108-qPCR-RCACellTest.png
qPCR curve
- 2016-11-09CellRCA-Gel1.png
Gel image 1
- 2016-11-09CellRCA-Gel2.png
Gel image 2
Controls Test[edit]
Testing the control samples that I have. I have used several positive controls and received no results when I should. So I'm testing the various control samples to see what comes out of it.
- qPCR
- Make master mix according to following recipes
- Add 47 uL master mix to each well
- Add 2 uL sample according to table in following section
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x35
- 72C 2 min
- 16C hold
- TBE Gel
- Mix 120 uL TBE, 30 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 | uL Forward Primer 10 uM) | uL Reverse Primer (10 uM) | uL 2X Kapa SYBR Fast Master Mix | uL nfH2O |
MM1 (4.1X) | p24/p4RC | 4.1 | 4.1 | 102.5 | 86.1 |
MM2 (4.1X) | p6/p12RC | 4.1 | 4.1 | 102.5 | 86.1 |
MM3 (3.1X) | p24/p12RC | 3.1 | 3.1 | 77.5 | 65.1 |
MM4 (3.1X) | p6/p4RC | 3.1 | 3.1 | 77.5 | 65.1 |
Results[edit]
- 20161108-qPCR-PosControlPrimers.png
qPCR curve, probe oligos
- 20161108-qPCR-AbOligoPrimers.png
qPCR curve-BSA Ab-congjugated oligos
- 2016-11-09-Primers-Gel1.png
Gel Image 1
- 2016-11-09-Primers-Gel2.png
Gel Image 2
qPCR Sample Table[edit]
Sample | Primers | Master Mix | Lane | Expected Product Size (bp) | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 10 uM Forward Primer | 10 uM Reverse Primer | H2O | Total Volume (uL) |
BSA-PA | p24/p4RC | MM1 | A1 | 44 | 2 | 25 | 1 | 1 | 21 | 50 |
BSA-PB | p6/p12RC | MM2 | A2 | 44 | 2 | 25 | 1 | 1 | 21 | 50 |
BSA-PA | p24/p12RC | MM3 | A3 | 0 | 2 | 25 | 1 | 1 | 21 | 50 |
BSA-PB | p6/p4RC | MM4 | A4 | 0 | 2 | 25 | 1 | 1 | 21 | 50 |
BSA Sample | p24/p4RC | MM1 | A5 | 44 | 2 | 25 | 1 | 1 | 21 | 50 |
BSA Sample | p6/p12RC | MM2 | A6 | 44 | 2 | 25 | 1 | 1 | 21 | 50 |
BSA Sample | p24/p12RC | MM3 | A7 | 0 | 2 | 25 | 1 | 1 | 21 | 50 |
BSA Sample | p6/p4RC | MM4 | A8 | 0 | 2 | 25 | 1 | 1 | 21 | 50 |
PCC-C1 | p24/p4RC | MM1 | H1 | 44 | 2 | 25 | 1 | 1 | 21 | 50 |
PCC-C1 | p6/p12RC | MM2 | H2 | 0 | 2 | 25 | 1 | 1 | 21 | 50 |
NTC | p24/p4RC | MM1 | H3 | 0 | 2 | 25 | 1 | 1 | 21 | 50 |
NTC | p24/p12RC | MM2 | H4 | 0 | 2 | 25 | 1 | 1 | 21 | 50 |
NTC | p6/p12RC | MM3 | H5 | 0 | 2 | 25 | 1 | 1 | 21 | 50 |
NTC | p6/p4RC | MM4 | H6 | 0 | 2 | 25 | 1 | 1 | 21 | 50 |