Daniel:Notebook/ComboLock/2016-11-30: Difference between revisions
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>Djacobse |
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{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | {| 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" | |- style="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center" | ||
| width=" | | width="85" height="30" | Sample | ||
| width=" | | width="125" | Condition | ||
| width="80" | RCA | |||
| width="85" | Lane | | width="85" | Lane | ||
| width="85" | AmpR Index | | width="85" | AmpR Index | ||
| width="85" | Sample Vol (uL) | | width="85" | Sample Vol (uL) | ||
| width=" | | width="86" | 2X Kapa SYBR qPCR MM | ||
| width=" | | width="84" | 10 uM Forward Primer | ||
| width=" | | width="83" | 10 uM Reverse Primer | ||
| width="85" | H2O | | width="85" | H2O | ||
| width=" | | width="86" | Total Volume (uL) | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
|style="font-weight:bold" height="15" | Sample 1A | |style="font-weight:bold" height="15" | Sample 1A | ||
| align="center" | Sample | | align="center" | Sample | ||
| align="center" | Y | |||
| align="center" | A1 | | align="center" | A1 | ||
| align="center" align="center" | 20 | | align="center" align="center" | 20 | ||
Line 143: | Line 145: | ||
|style="font-weight:bold" height="15" | Sample 1B | |style="font-weight:bold" height="15" | Sample 1B | ||
| align="center" | Sample | | align="center" | Sample | ||
| align="center" | Y | |||
| align="center" | A2 | | align="center" | A2 | ||
| align="center" align="center" | 20 | | align="center" align="center" | 20 | ||
Line 155: | Line 158: | ||
|style="font-weight:bold" height="15" | Sample 2A | |style="font-weight:bold" height="15" | Sample 2A | ||
| align="center" | No Template | | align="center" | No Template | ||
| align="center" | Y | |||
| align="center" | A3 | | align="center" | A3 | ||
| align="center" align="center" | 21 | | align="center" align="center" | 21 | ||
Line 167: | Line 171: | ||
|style="font-weight:bold" height="15" valign="bottom" | Sample 2B | |style="font-weight:bold" height="15" valign="bottom" | Sample 2B | ||
| align="center" valign="bottom" | No Template | | align="center" valign="bottom" | No Template | ||
| align="center" valign="bottom" | Y | |||
| align="center" | A4 | | align="center" | A4 | ||
| align="center" align="center" | 21 | | align="center" align="center" | 21 | ||
Line 179: | Line 184: | ||
|style="font-weight:bold" height="15" | Sample 3A | |style="font-weight:bold" height="15" | Sample 3A | ||
| align="center" | No Phosphate | | align="center" | No Phosphate | ||
| align="center" | Y | |||
| align="center" | A5 | | align="center" | A5 | ||
| align="center" align="center" | 22 | | align="center" align="center" | 22 | ||
Line 191: | Line 197: | ||
|style="font-weight:bold" height="15" | Sample 3B | |style="font-weight:bold" height="15" | Sample 3B | ||
| align="center" | No Phosphate | | align="center" | No Phosphate | ||
| align="center" | Y | |||
| align="center" | A6 | | align="center" | A6 | ||
| align="center" align="center" | 22 | | align="center" align="center" | 22 | ||
Line 203: | Line 210: | ||
|style="font-weight:bold" height="15" | Sample 4A | |style="font-weight:bold" height="15" | Sample 4A | ||
| align="center" | No C Probe | | align="center" | No C Probe | ||
| align="center" | Y | |||
| align="center" | A7 | | align="center" | A7 | ||
| align="center" align="center" | 23 | | align="center" align="center" | 23 | ||
Line 215: | Line 223: | ||
|style="font-weight:bold" height="15" | Sample 4B | |style="font-weight:bold" height="15" | Sample 4B | ||
| align="center" | No C Probe | | align="center" | No C Probe | ||
| align="center" | Y | |||
| align="center" | A8 | | align="center" | A8 | ||
| align="center" align="center" | 23 | | 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 1A | |||
| align="center" | Sample | |||
| align="center" | N | |||
| align="center" | A1 | |||
| 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:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 1B | |||
| align="center" | Sample | |||
| 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="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 2A | |||
| align="center" | No Template | |||
| align="center" | N | |||
| align="center" | A3 | |||
| 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:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Sample 2B | |||
| align="center" valign="bottom" | No Template | |||
| align="center" valign="bottom" | N | |||
| align="center" | A4 | |||
| 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 3A | |||
| align="center" | No Phosphate | |||
| align="center" | N | |||
| align="center" | A5 | |||
| 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:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 3B | |||
| align="center" | No Phosphate | |||
| align="center" | N | |||
| align="center" | A6 | |||
| 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 4A | |||
| align="center" | No C Probe | |||
| align="center" | N | |||
| align="center" | A7 | |||
| 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:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 4B | |||
| align="center" | No C Probe | |||
| align="center" | N | |||
| align="center" | A8 | |||
| align="center" align="center" | 27 | |||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
| align="center" align="center" | 25 | | align="center" align="center" | 25 | ||
Line 227: | Line 340: | ||
|style="font-weight:bold" height="15" valign="bottom" | NTC | |style="font-weight:bold" height="15" valign="bottom" | NTC | ||
| align="center" valign="bottom" | qPCR Primers Only | | align="center" valign="bottom" | qPCR Primers Only | ||
| align="center" valign="bottom" | NA | |||
| align="center" | H1 | | align="center" | H1 | ||
| align="center" align="center" | | | align="center" align="center" | 28 | ||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
| align="center" align="center" | 25 | | align="center" align="center" | 25 | ||
Line 237: | Line 351: | ||
|} | |} | ||
<li>qPCR Cycles</li> | <li>qPCR Cycles</li> | ||
<ol type="a"> | <ol type="a"> |
Revision as of 21:06, 30 November 2016
Extended Latch Test (Started Daniel:Notebook/ComboLock/2016-11-29)
Sample Matrix
Sample | Condition |
Sample 1 (AB) | Normal |
Sample 2 (AB) | No Template |
Sample 3 (AB) | No C Probe |
Sample 4 (AB) | No Kinase |
Protocol
- Exonuclease Digestion
- Combine 15 uL of Exonuclease I (20U/uL) and 3 uL Exonuclease III (100U/uL)
- Add 2 uL of mix to each reaction
- Incubate for 30 minutes to 2 hours at 37C
- Heat kill by incubating for 5 minutes at 94C
- Rolling Circle Amplification
- Prepare 9X master mix according to table below
- Add 15 uL master mix to each tube
- Add 5 uL sample to correct reaction tubes
- Incubate at 37C for 3 hours
- Heat kill by incubating at 65C for 10 minutes
- qPCR
- Make master mix according to following recipe
- 191.1 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 200 uL TBE, 50 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 | Stock Concentration | Final Conc/Amt | uL Added in 20 uL Rxn | Master Mix (9X) |
Template | NA | NA | 5 | 45 |
RCA Primer (RCA-CL-LinkerLRC-v01) | 10 uM | 2 uM | 4 | 36 |
dNTP | 1 mM | 50 uM | 1 | 9 |
10X Buffer | 10X | 1X | 2 | 18 |
Phi29 (NEB) | 10 U/uL | 10 U | 1 | 9 |
nfH2O | NA | NA | 7 | 63 |
Total | 20 | 180 |
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 | Sample | Y | A1 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1B | Sample | Y | A2 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2A | No Template | Y | A3 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2B | No Template | Y | A4 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3A | No Phosphate | Y | A5 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3B | No Phosphate | Y | A6 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4A | No C Probe | Y | A7 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4B | No C Probe | Y | A8 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1A | Sample | N | A1 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1B | Sample | N | A2 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2A | No Template | N | A3 | 25 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2B | No Template | N | A4 | 25 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3A | No Phosphate | N | A5 | 26 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3B | No Phosphate | N | A6 | 26 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4A | No C Probe | N | A7 | 27 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4B | No C Probe | N | A8 | 27 | 2 | 25 | 1 | 1 | 21 | 50 |
NTC | qPCR Primers Only | NA | H1 | 28 | 2 | 25 | 1 | 1 | 23 | 50 |