Daniel:Notebook/PosSequencing/2017-6-20: Difference between revisions
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>Djacobse (Created page with "=Emulsion PCR Test 5 (Started Yesterday)= Back to Calendar ==Protocol== <ol start="6"> <li>Pre...") |
>Djacobse |
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<li>Make the following master mixes</li> | <li>Make the following master mixes</li> | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#B1A0C7;font-size:12pt;font-weight:bold" align="center" | |||
| width="140" height="30" | Reagent | |||
| width="95" | uL in Single Rxn | |||
| width="95" | Master Mix P2 (6.2X) | |||
| width="95" | Master Mix BioU (6.2X) | |||
| width="95" | Master Mix Dual (6.2X) | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Primer 2 (10 uM) | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 6.2 | |||
| align="center" align="center" valign="bottom" | 0 | |||
| align="center" align="center" valign="bottom" | 6.2 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | BioU Primer (10 uM) | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 0 | |||
| align="center" align="center" valign="bottom" | 6.2 | |||
| align="center" align="center" valign="bottom" | 6.2 | |||
|- style="font-size:12pt" | |||
| height="30" valign="bottom" | 2X Kapa SYBR Master Mix | |||
| align="center" align="center" valign="bottom" | 20 | |||
| align="center" align="center" valign="bottom" | 124 | |||
| align="center" align="center" valign="bottom" | 124 | |||
| align="center" align="center" valign="bottom" | 124 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | nfH2O | |||
| align="center" align="center" valign="bottom" | 14 | |||
| align="center" align="center" valign="bottom" | 86.8 | |||
| align="center" align="center" valign="bottom" | 86.8 | |||
| align="center" align="center" valign="bottom" | 80.6 | |||
|- style="font-size:12pt" | |||
| height="30" valign="bottom" | Total* (+ 5 uL sample=40) | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 35 | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 217 | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 217 | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 217 | |||
|} | |||
<li>Aliquot 35 uL master mix to appropriate lanes</li> | <li>Aliquot 35 uL master mix to appropriate lanes</li> | ||
<li>Add 2 uL sample according to plate layout below</li> | <li>Add 2 uL sample according to plate layout below</li> | ||
[[Image:|522x348px]] | [[Image:PlateLayout-SinglePrimer-20170620-emPCRTest5.png|522x348px]] | ||
<li>Run the following thermocycler program</li> | <li>Run the following thermocycler program</li> |
Revision as of 15:59, 20 June 2017
Emulsion PCR Test 5 (Started Yesterday)
Protocol
- Prepare Water saturated compounds
- Mix 3 mL diethyl ether and 3 mL ddH2O in a falcon tube; allow to settle, extract top (ether) phase
- Mix 3 mL ethyl acetate and 3 mL ddH2O in a falcon tube; allow to settle, extract top (acetate) phase
- Emulsion Breaking
- With a 200 uL pipette add 100 uL diethyl ether to each sample; save the tip
- Adjust tip to 200 and using same tip pool samples in a new 1.5 mL tube
- Vortex the pooled sample in the 1.5 mL tube for 30 seconds
- Spin down the sample 13,000 rpm for 5 minutes
- Take off oil-DE mix (top layer) but do not disrupt the oil/aqueous interface
- Add 1000 uL ethyl acetate and vortex for 30 seconds
- Spin down sample at 13,000rpm for 3 minutes
- Remove ethyl acetate without disturbing the pellet
- Add 1000 uL diethyl ether and vortex for 30 seconds
- Spin down sample at 13,000rpm for 3 minutes
- Remove DE without disturbing the aqueous phase
- Add 500 uL 4X SSC wash buffer to 1.5 mL tube and vortex for 30 seconds
- Spin down sample at 13,000rpm for 1 minute
- Remove all but 40uL wash buffer
- Repeat steps 11 to 15 one more time (j to o)
- On last wash, take 15 uL (of 100) for qPCR
- Strand Separation (DNA Denaturation)
- Add 500 uL KOH buffer (pH 12.0)
- Spin down sample at 13,000rpm for 1 minute
- Remove all but 40uL buffer
- Repeat KOH steps A-C once more
- Add 500 uL 4X SSC buffer (pH 12.0)
- Spin down sample at 13,000rpm for 1 minute
- Remove all but 10uL buffer
- Resuspend in 80 uL 2X SSC buffer
- Use this for final step of qPCR
- Single primer qPCR
- Make the following master mixes
- Aliquot 35 uL master mix to appropriate lanes
- Add 2 uL sample according to plate layout below File:PlateLayout-SinglePrimer-20170620-emPCRTest5.png
- Run the following thermocycler program
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x30
- 72C 2 min
- 16C hold
******
Reagent | uL in Single Rxn | Master Mix P2 (6.2X) | Master Mix BioU (6.2X) | Master Mix Dual (6.2X) |
Primer 2 (10 uM) | 1 | 6.2 | 0 | 6.2 |
BioU Primer (10 uM) | 1 | 0 | 6.2 | 6.2 |
2X Kapa SYBR Master Mix | 20 | 124 | 124 | 124 |
nfH2O | 14 | 86.8 | 86.8 | 80.6 |
Total* (+ 5 uL sample=40) | 35 | 217 | 217 | 217 |
Results
- PlateLayout-SinglePrimer-DualBiotin-20170615.png
Plate layout
- Singleprimer-20170615-dualbiotin-plateCTs.png
Plate CTs
- Singleprimer-20170615-dualbiotin-plateCTs-normed.png
Plate CTs-normed
- Singleprimer-20170615-dualbiotin-rawcurves.png
Raw curves