Daniel:Notebook/PosSequencing/2017-6-29: Difference between revisions
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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:#B1A0C7;font-size:12pt;font-weight:bold" align="center" | |- style="background-color:#B1A0C7;font-size:12pt;font-weight:bold" align="center" | ||
| width=" | | width="190" 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" | 16 | | align="center" align="center" valign="bottom" | 16 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 195.2 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 195.2 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 213 | ||
|- 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" | 451.4 | ||
|style="font-weight:bold" align="center" align="center" valign="bottom" | | |style="font-weight:bold" align="center" align="center" valign="bottom" | 451.4 | ||
|style="font-weight:bold" align="center" align="center" valign="bottom" | | |style="font-weight:bold" align="center" align="center" valign="bottom" | 525.4 | ||
|} | |} |
Revision as of 16:09, 29 June 2017
emPCR 7-Strand Separation (Started Yesterday)
Protocol (From Yesterday)
- 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
- Aliquot 35 uL master mix to appropriate lanes
- Add 2 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 | 16 | 195.2 | 195.2 | 213 |
Total* (+ 5 uL sample=40) | 35 | 451.4 | 451.4 | 525.4 |