Daniel:Notebook/ComboLock/2017-2-3: Difference between revisions
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>Djacobse |
>Djacobse |
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| width="65" | uL Added | | width="65" | uL Added | ||
| width="105" | Master Mix (8.2X) | | width="105" | Master Mix (8.2X) | ||
| width="105" | Master Mix (4.2X) | |||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | Template | | height="15" valign="bottom" | Template | ||
| align="center" align="center" valign="bottom" | 5 | | align="center" align="center" valign="bottom" | 5 | ||
| align="center" valign="bottom" | NA | |||
| align="center" valign="bottom" | NA | | align="center" valign="bottom" | NA | ||
Line 32: | Line 34: | ||
| align="center" align="center" valign="bottom" | 2.5 | | align="center" align="center" valign="bottom" | 2.5 | ||
| align="center" align="center" valign="bottom" | 20.5 | | align="center" align="center" valign="bottom" | 20.5 | ||
| align="center" align="center" valign="bottom" | 10.5 | |||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
Line 37: | Line 40: | ||
| align="center" align="center" valign="bottom" | 5 | | align="center" align="center" valign="bottom" | 5 | ||
| align="center" align="center" valign="bottom" | 41 | | align="center" align="center" valign="bottom" | 41 | ||
| align="center" align="center" valign="bottom" | 21 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
Line 42: | Line 46: | ||
| align="center" align="center" valign="bottom" | 2 | | align="center" align="center" valign="bottom" | 2 | ||
| align="center" align="center" valign="bottom" | 16.4 | | align="center" align="center" valign="bottom" | 16.4 | ||
| align="center" align="center" valign="bottom" | 8.4 | |||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | Phi29 | | height="15" valign="bottom" | Phi29 | ||
| align="center" align="center" valign="bottom" | 1 | | align="center" align="center" valign="bottom" | 1 | ||
| align="center" align="center" valign="bottom" | 0 | |||
| align="center" align="center" valign="bottom" | 0 | | align="center" align="center" valign="bottom" | 0 | ||
Line 52: | Line 58: | ||
| align="center" align="center" valign="bottom" | 0.4 | | align="center" align="center" valign="bottom" | 0.4 | ||
| align="center" align="center" valign="bottom" | 3.28 | | align="center" align="center" valign="bottom" | 3.28 | ||
| align="center" align="center" valign="bottom" | 0 | |||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
Line 57: | Line 64: | ||
| align="center" align="center" valign="bottom" | 4.1 | | align="center" align="center" valign="bottom" | 4.1 | ||
| align="center" align="center" valign="bottom" | 33.62 | | align="center" align="center" valign="bottom" | 33.62 | ||
| align="center" align="center" valign="bottom" | 18.9 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | Total | | height="15" valign="bottom" | Total | ||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 20 | |style="font-weight:bold" align="center" align="center" valign="bottom" | 20 | ||
|style="font-weight:bold" align="center" align="center" valign="bottom" | | |style="font-weight:bold" align="center" align="center" valign="bottom" | 114.8 | ||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 58.8 | |||
|} | |} | ||
<li>Add 14 uL master mix to each tube</li> | <li>Add 14 uL master mix to each tube</li> | ||
<li>Add sample to new reaction tubes</li> | <li>Add sample and 1 uL appropriate enzyme to new reaction tubes</li> | ||
<li>Incubate at 37C for 3 hours</li> | <li>Incubate at 37C for 3 hours</li> | ||
<li>Incubate at 65C for 10 minutes;Hold at 10C until next step</li></ol></ol> | <li>Incubate at 65C for 10 minutes;Hold at 10C until next step</li></ol></ol> |
Revision as of 17:27, 3 February 2017
Version 2 Oligos Round 2 (Started yesterday)
Protocol-Part 4-RCA
- 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 hour at 37C
- Heat kill by incubating for 5 min at 95C
- Rolling Circle Amplification
- Prepare 8.2X Master mix according to table below
- Add 14 uL master mix to each tube
- Add sample and 1 uL appropriate enzyme to new reaction tubes
- Incubate at 37C for 3 hours
- Incubate at 65C for 10 minutes;Hold at 10C until next step
Reagent | uL Added | Master Mix (8.2X) | Master Mix (4.2X) |
Template | 5 | NA | NA |
RCA Primer (10 uM) | 2.5 | 20.5 | 10.5 |
dNTP (1 mM) | 5 | 41 | 21 |
10X Buffer | 2 | 16.4 | 8.4 |
Phi29 | 1 | 0 | 0 |
BSA (10 mg/mL) | 0.4 | 3.28 | 0 |
nfH2O | 4.1 | 33.62 | 18.9 |
Total | 20 | 114.8 | 58.8 |
Protocol Part 5-qPCR and Gel
- 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 in the following section
- 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