Daniel:Notebook/ComboLock/2017-1-24: Difference between revisions
Jump to navigation
Jump to search
>Djacobse |
>Djacobse |
||
Line 107: | Line 107: | ||
{| 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="120" height="45" | Sample | ||
| width=" | | width="100" | Product Amt | ||
| width="60" | Pre/Post RCA | | width="60" | Pre/Post RCA | ||
| width=" | | width="120" | Primer Pair | ||
| width="65" | Lane | | width="65" | Lane | ||
| width="65" | AmpR Index | | width="65" | AmpR Index |
Revision as of 21:38, 24 January 2017
Circularization Test (Started Yesterday)
Protocol-Part 2
- Exonuclease Digestion
- Mix 15 uL Exo I (20U/uL) and 3 uL ExoIII (100U/uL)
- Add 2 uL to each sample, mix by swirling pipette tip
- Incubate for 1.5 hours at 37C
- Heat kill by incubating for 5 min at 95C
- Rolling Circle Amplification
- Prepare 7X Master mix according to table below
- Add 15 uL master mix to each tube
- 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 until next step
- 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
- 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 80 uL TBE, 20 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 (8.5X) |
Template | 5 | NA |
RCA Primer (10 uM) | 2.5 | 21.25 |
dNTP (1 mM) | 0.8 | 6.8 |
10X Buffer | 2 | 17 |
Phi29 | 1 | 8.5 |
nfH2O | 8.7 | 73.95 |
Total | 20 | 127.5 |
Protocol-Part 3
qPCR Sample Table
Sample | Product Amt | Pre/Post RCA | Primer Pair | Lane | AmpR Index | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 10 uM Forward Primer | 10 uM Reverse Primer | H2O | Total Volume (uL) | Master Mix |
Sample 0A | 1pmol | Pre | AmpF/AmpR | A1 | 21 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 0B | 1pmol | Pre | AmpF/AmpR | A2 | 21 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 2A | 10 fmol | Pre | AmpF/AmpR | A3 | 22 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 2B | 10 fmol | Pre | AmpF/AmpR | A4 | 22 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 4A | 100 amol | Pre | AmpF/AmpR | A5 | 23 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 4B | 100 amol | Pre | AmpF/AmpR | A6 | 23 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 6A | 1 amol | Pre | AmpF/AmpR | A7 | 24 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 6B | 1 amol | Pre | AmpF/AmpR | A8 | 24 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 0AX | 1pmol | Post | AmpF/AmpR | H1 | 25 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 0BX | 1pmol | Post | AmpF/AmpR | H2 | 25 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 2AX | 10 fmol | Post | AmpF/AmpR | H3 | 26 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 2BX | 10 fmol | Post | AmpF/AmpR | H4 | 26 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 4AX | 100 amol | Post | AmpF/AmpR | H5 | 27 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 4BX | 100 amol | Post | AmpF/AmpR | H6 | 27 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 6AX | 1 amol | Post | AmpF/AmpR | H7 | 28 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 6BX | 1 amol | Post | AmpF/AmpR | H8 | 28 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 0A | 1pmol | Pre | primer6RC/AmpR | C1 | 29 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 0B | 1pmol | Pre | primer6RC/AmpR | C2 | 29 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 6A | 1 amol | Pre | primer6RC/AmpR | C3 | 29 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 6B | 1 amol | Pre | primer6RC/AmpR | C4 | 29 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 0AX | 1pmol | Post | primer6RC/AmpR | C5 | 29 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 0BX | 1pmol | Post | primer6RC/AmpR | C6 | 29 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 6AX | 1 amol | Post | primer6RC/AmpR | C7 | 29 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 6BX | 1 amol | Post | primer6RC/AmpR | C8 | 29 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
NTC | qPCR Primers Only | NA | AmpF/AmpR | F1 | 30 | 2 | 25 | 1 | 1 | 23 | 50 | MM1 |
NTC | qPCR Primers Only | NA | primer6RC/AmpR | F2 | 30 | 2 | 25 | 1 | 1 | 23 | 50 | MM2 |