Daniel:Notebook/ComboLock/2016-11-1: Difference between revisions
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===qPCR Sample Table=== | ===qPCR Sample Table=== | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center" | |||
| width="105" height="45" | Sample | |||
| width="65" | Condition | |||
| width="50" | RCA? | |||
| width="65" | Lane | |||
| width="65" | AmpR Index | |||
| width="65" | Sample Vol (uL) | |||
| width="85" | 2X Kapa SYBR qPCR MM | |||
| width="85" | 10 uM Forward Primer | |||
| width="85" | 10 uM Reverse Primer | |||
| width="65" | H2O | |||
| width="65" | Total Volume (uL) | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 1A | |||
|style="font-weight:bold" align="center" | K-ON-Y | |||
| align="center" | N | |||
| align="center" | A1 | |||
| align="center" align="center" | 20 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 1B | |||
|style="font-weight:bold" align="center" | K-ON-Y | |||
| align="center" | N | |||
| align="center" | B1 | |||
| align="center" align="center" | 20 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 2A | |||
|style="font-weight:bold" align="center" | K-ON-N | |||
| align="center" | N | |||
| align="center" | C1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Sample 2B | |||
|style="font-weight:bold" align="center" valign="bottom" | K-ON-N | |||
| align="center" | N | |||
| align="center" | D1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 3A | |||
|style="font-weight:bold" align="center" | K-2H-Y | |||
| align="center" | N | |||
| align="center" | E1 | |||
| align="center" align="center" | 22 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 3B | |||
|style="font-weight:bold" align="center" | K-2H-Y | |||
| align="center" | N | |||
| align="center" | F1 | |||
| align="center" align="center" | 22 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 4A | |||
|style="font-weight:bold" align="center" | K-2H-N | |||
| align="center" | N | |||
| align="center" | G1 | |||
| align="center" align="center" | 23 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Sample 4B | |||
|style="font-weight:bold" align="center" valign="bottom" | K-2H-N | |||
| align="center" | N | |||
| align="center" | H1 | |||
| align="center" align="center" | 23 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 5A | |||
|style="font-weight:bold" align="center" | P-ON-Y | |||
| align="center" | N | |||
| align="center" | A2 | |||
| align="center" align="center" | 24 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#DA9694;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 5B | |||
|style="font-weight:bold" align="center" | P-ON-Y | |||
| align="center" | N | |||
| align="center" | B2 | |||
| align="center" align="center" | 24 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 6A | |||
|style="font-weight:bold" align="center" | P-ON-N | |||
| align="center" | N | |||
| align="center" | C2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#DA9694;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Sample 6B | |||
|style="font-weight:bold" align="center" valign="bottom" | P-ON-N | |||
| align="center" | N | |||
| align="center" | D2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 7A | |||
|style="font-weight:bold" align="center" | P-2H-Y | |||
| align="center" | N | |||
| align="center" | E2 | |||
| align="center" align="center" | 26 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#DA9694;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 7B | |||
|style="font-weight:bold" align="center" | P-2H-Y | |||
| align="center" | N | |||
| align="center" | F2 | |||
| align="center" align="center" | 26 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 8A | |||
|style="font-weight:bold" align="center" | P-2H-N | |||
| align="center" | N | |||
| align="center" | G2 | |||
| align="center" align="center" | 27 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#DA9694;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Sample 8B | |||
|style="font-weight:bold" align="center" valign="bottom" | P-2H-N | |||
| align="center" | N | |||
| align="center" | H2 | |||
| align="center" align="center" | 27 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 1A | |||
|style="font-weight:bold" align="center" | K-ON-Y | |||
| align="center" | Y | |||
| align="center" | A12 | |||
| align="center" align="center" | 28 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#C4D79B;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 1B | |||
|style="font-weight:bold" align="center" | K-ON-Y | |||
| align="center" | Y | |||
| align="center" | B12 | |||
| align="center" align="center" | 28 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 3A | |||
|style="font-weight:bold" align="center" | K-ON-Y | |||
| align="center" | Y | |||
| align="center" | C12 | |||
| align="center" align="center" | 29 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#C4D79B;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Sample 3B | |||
|style="font-weight:bold" align="center" | K-ON-Y | |||
| align="center" | Y | |||
| align="center" | D12 | |||
| align="center" align="center" | 29 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 5A | |||
|style="font-weight:bold" align="center" | P-ON-Y | |||
| align="center" | Y | |||
| align="center" | E12 | |||
| align="center" align="center" | 30 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#C4D79B;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 5B | |||
|style="font-weight:bold" align="center" | P-ON-Y | |||
| align="center" | Y | |||
| align="center" | F12 | |||
| align="center" align="center" | 30 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 7A | |||
|style="font-weight:bold" align="center" | P-ON-Y | |||
| align="center" | Y | |||
| align="center" | G12 | |||
| align="center" align="center" | 31 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#C4D79B;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Sample 7B | |||
|style="font-weight:bold" align="center" | P-ON-Y | |||
| align="center" | Y | |||
| align="center" | H12 | |||
| align="center" align="center" | 31 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | NTC | |||
|style="font-weight:bold" align="center" | NA | |||
| align="center" | N | |||
| align="center" | A4 | |||
| align="center" align="center" | 33 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|} | |||
===Results=== | ===Results=== |
Revision as of 20:45, 1 November 2016
Circularization Optimization Testing
Sample Matrix
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
- 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
- 54.25 uL nfH2O
- 77.5 uL SYBR Fast
- 1.55 uL AmpF
- Add 43 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 48 uL TBE, 12 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
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 | B1 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2A | K-ON-N | N | C1 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2B | K-ON-N | N | D1 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3A | K-2H-Y | N | E1 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3B | K-2H-Y | N | F1 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4A | K-2H-N | N | G1 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4B | K-2H-N | N | H1 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 5A | P-ON-Y | N | A2 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 5B | P-ON-Y | N | B2 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 6A | P-ON-N | N | C2 | 25 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 6B | P-ON-N | N | D2 | 25 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 7A | P-2H-Y | N | E2 | 26 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 7B | P-2H-Y | N | F2 | 26 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 8A | P-2H-N | N | G2 | 27 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 8B | P-2H-N | N | H2 | 27 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1A | K-ON-Y | Y | A12 | 28 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1B | K-ON-Y | Y | B12 | 28 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3A | K-ON-Y | Y | C12 | 29 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3B | K-ON-Y | Y | D12 | 29 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 5A | P-ON-Y | Y | E12 | 30 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 5B | P-ON-Y | Y | F12 | 30 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 7A | P-ON-Y | Y | G12 | 31 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 7B | P-ON-Y | Y | H12 | 31 | 2 | 25 | 1 | 1 | 21 | 50 |
NTC | NA | N | A4 | 33 | 2 | 25 | 1 | 1 | 21 | 50 |