Daniel:Notebook/PosSequencing/2017-6-29: Difference between revisions
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>Djacobse |
>Djacobse m (→qPCR Round 2) |
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(11 intermediate revisions by the same user not shown) | |||
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| width="210" height="30" | Reagent | | width="210" height="30" | Reagent | ||
| width="95" | uL in Single Rxn | | width="95" | uL in Single Rxn | ||
| width="95" | Master Mix P2 ( | | width="95" | Master Mix P2 (12.2X) | ||
| width=" | | width="110" | Master Mix BioU (12.2X) | ||
| width=" | | width="110" | Master Mix Dual (14.2X) | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | Primer 2 (10 uM) | | height="15" valign="bottom" | Primer 2 (10 uM) | ||
| align="center" align="center" valign="bottom" | 1 | | align="center" align="center" valign="bottom" | 1 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 12.2 | ||
| align="center" align="center" valign="bottom" | 0 | | align="center" align="center" valign="bottom" | 0 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 14.2 | ||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
Line 41: | Line 41: | ||
| 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" | | | align="center" align="center" valign="bottom" | 12.2 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 14.2 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | 2X Kapa SYBR Master Mix | | height="15" valign="bottom" | 2X Kapa SYBR Master Mix | ||
| align="center" align="center" valign="bottom" | 20 | | align="center" align="center" valign="bottom" | 20 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 244 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 244 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 284 | ||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | nfH2O | | height="15" valign="bottom" | nfH2O | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 9 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 109.8 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 109.8 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 113.6 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | Total* (+ 5 uL sample=40) | | height="15" 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" | 35 | ||
|style="font-weight:bold" align="center" align="center" valign="bottom" | | |style="font-weight:bold" align="center" align="center" valign="bottom" | 366 | ||
|style="font-weight:bold" align="center" align="center" valign="bottom" | | |style="font-weight:bold" align="center" align="center" valign="bottom" | 366 | ||
|style="font-weight:bold" align="center" align="center" valign="bottom" | | |style="font-weight:bold" align="center" align="center" valign="bottom" | 426 | ||
|} | |} | ||
<li>Aliquot | *Notes: positive controls are 5 uL sample/5 uL nfH<sub>2</sub>O | ||
<li>Aliquot 30 uL master mix to appropriate lanes</li> | |||
<li>Add 10 uL sample according to plate layout below</li> | |||
[[Image:PlateLayout20170629-emPCR7-SinglePrimer.png|522x348px]] | |||
<li>Run the following thermocycler program</li> | |||
<ol type="a"> | |||
<li>95C 3 min</li> | |||
<li>95C 3 sec</li> | |||
<li>45C 30 sec</li> | |||
<li>72C 20 sec</li> | |||
<li>plate read</li> | |||
<li>goto b x90</li> | |||
<li>72C 2 min</li> | |||
<li>16C hold</li> | |||
</ol></ol> | |||
</ol> | |||
==Results== | |||
<gallery perrow=2 heights=250px widths=350px mode=packed-hover> | |||
File:PlateLayout20170629-emPCR7-SinglePrimer.png|Plate Layout | |||
File:EmPCR7-20170629-singleprimer-plateCTs-normed.png|Plate CTs | |||
File:EmPCR7-20170629-singleprimer-rawcurves.png|Raw curves | |||
</gallery> | |||
==qPCR Round 2== | |||
This is to double check the results of the previous experiment, which show little product for any the samples. Specifically,the post-emulsion breaking samples showed no discernible product, even though there was product in the pH 10 sample post-alkaline denaturation. | |||
<ol> | |||
<li>qPCR</li> | |||
<ol type="A"> | |||
<li>Make the following 8.2X master mix</li> | |||
<ol type="a"> | |||
<li>8.2 uL 10 uM primer2</li> | |||
<li>8.2 uL 10 uM 2BiotinUracil</li> | |||
<li>164 uL 10 uM 2X Kapa SYBR Fast master mix</li> | |||
<li>131.2 uL 10 uM nfH<sub>2</sub>O</li> | |||
</ol> | |||
<li>Aliquot 38 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:PlateLayout20170629-emPCR7- | [[Image:PlateLayout20170629-emPCR7-aqueousfractions.png|522x348px]] | ||
<li>Run the following thermocycler program</li> | <li>Run the following thermocycler program</li> | ||
Line 84: | Line 126: | ||
</ol></ol> | </ol></ol> | ||
</ol> | </ol> | ||
===Results=== | |||
<gallery perrow=2 heights=250px widths=350px mode=packed-hover> | |||
File:PlateLayout20170629-emPCR7-aqueousfractions.png|Plate Layout | |||
File:EmPCR7-20170629-emBreak-plateCTs-normed.png|Plate CTs | |||
File:EmPCR7-20170629-emBreak-rawcurves.png|Raw curves | |||
File:2017-06-30-PosSeq-emPCR7-aqueousBeads.png|Gel image | |||
</gallery> | |||
[[Category:20170628]] [[Category:PosSeq]] |
Latest revision as of 20:06, 30 June 2017
emPCR 7-Strand Separation (Started Yesterday)[edit]
Protocol (From Yesterday)[edit]
- Strand Separation (DNA Denaturation)
- Split each sample (1 pM and 100 pM) into 4 parts-> 25 uL each-> put them in 1.5mL tubes
- Add 200 uL KOH buffer to each sample(pH 12.0, 11.5, 11.0, 10.0); denaturation pH of DNA should be around 11.3
- Spin down sample at 13,000rpm for 1 minute
- Remove all but 40uL buffer
- Repeat KOH steps A-C once more
- Add 200 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
- Single primer qPCR
- Make the following master mixes
- Notes: positive controls are 5 uL sample/5 uL nfH2O
- Aliquot 30 uL master mix to appropriate lanes
- Add 10 uL sample according to plate layout below File:PlateLayout20170629-emPCR7-SinglePrimer.png
- Run the following thermocycler program
- 95C 3 min
- 95C 3 sec
- 45C 30 sec
- 72C 20 sec
- plate read
- goto b x90
- 72C 2 min
- 16C hold
Reagent | uL in Single Rxn | Master Mix P2 (12.2X) | Master Mix BioU (12.2X) | Master Mix Dual (14.2X) |
Primer 2 (10 uM) | 1 | 12.2 | 0 | 14.2 |
BioU Primer (10 uM) | 1 | 0 | 12.2 | 14.2 |
2X Kapa SYBR Master Mix | 20 | 244 | 244 | 284 |
nfH2O | 9 | 109.8 | 109.8 | 113.6 |
Total* (+ 5 uL sample=40) | 35 | 366 | 366 | 426 |
Results[edit]
- PlateLayout20170629-emPCR7-SinglePrimer.png
Plate Layout
- EmPCR7-20170629-singleprimer-plateCTs-normed.png
Plate CTs
- EmPCR7-20170629-singleprimer-rawcurves.png
Raw curves
qPCR Round 2[edit]
This is to double check the results of the previous experiment, which show little product for any the samples. Specifically,the post-emulsion breaking samples showed no discernible product, even though there was product in the pH 10 sample post-alkaline denaturation.
- qPCR
- Make the following 8.2X master mix
- 8.2 uL 10 uM primer2
- 8.2 uL 10 uM 2BiotinUracil
- 164 uL 10 uM 2X Kapa SYBR Fast master mix
- 131.2 uL 10 uM nfH2O
- Aliquot 38 uL master mix to appropriate lanes
- Add 2 uL sample according to plate layout below File:PlateLayout20170629-emPCR7-aqueousfractions.png
- Run the following thermocycler program
- 95C 3 min
- 95C 3 sec
- 45C 30 sec
- 72C 20 sec
- plate read
- goto b x90
- 72C 2 min
- 16C hold
Results[edit]
- PlateLayout20170629-emPCR7-aqueousfractions.png
Plate Layout
- EmPCR7-20170629-emBreak-plateCTs-normed.png
Plate CTs
- EmPCR7-20170629-emBreak-rawcurves.png
Raw curves
- 2017-06-30-PosSeq-emPCR7-aqueousBeads.png
Gel image