Daniel:Notebook/PosSequencing/2017-5-24: Difference between revisions
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>Djacobse |
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<li>Add 50 uL (dual) to the appropriate lanes</li> | <li>Add 50 uL (dual) to the appropriate lanes</li> | ||
[[ | [[Image:PlateLayout-PosSeq-BarcodeOligoProduction-20170524.png|400x300px]] | ||
<li>qPCR Cycles</li> | <li>qPCR Cycles</li> | ||
Line 111: | Line 111: | ||
<li>16C hold</li> | <li>16C hold</li> | ||
</ol></ol> | </ol></ol> | ||
==Results== | |||
<gallery perrow=3 heights=200px widths=300px mode=packed-hover> | |||
File:PlateLayout-PosSeq-BarcodeOligoProduction-20170524.png|Plate layout | |||
File:BarcodeProduction-20170524-platelayout.png|plate CTs | |||
File:BarcodeProduction-20170524-rawcurves.png|raw curves | |||
</gallery> | |||
[[Category:20170524]] [[Category:OligoPrep]] [[Category:PosSeq]] |
Latest revision as of 18:21, 30 May 2017
Barcode Oligo Production[edit]
Making more double stranded barcode oligo since I'm running out. I'll make both EcoRI and Uracil versions.
Protocol[edit]
- qPCR
- Make the following master mixes
- Add 50 uL (dual) to the appropriate lanes File:PlateLayout-PosSeq-BarcodeOligoProduction-20170524.png
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x30
- 72C 2 min
- 16C hold
- PlateLayout-PosSeq-BarcodeOligoProduction-20170524.png
Plate layout
- BarcodeProduction-20170524-platelayout.png
plate CTs
- BarcodeProduction-20170524-rawcurves.png
raw curves
Reagent | uL Single rxn | Master Mix Eco1 (8.2X) | Master Mix Eco2 (8.2X) | Master Mix Eco3 (8.2X) | Master Mix BioU1 (8.2X) | Master Mix BioU2 (8.2X) | Master Mix BioU3 (8.2X) |
Biotin-Uracil Primer (10 uM) | 1 | 0 | 0 | 0 | 8.2 | 8.2 | 8.2 |
Biotin-EcoRI Primer (10 uM) | 1 | 8.2 | 8.2 | 8.2 | 0 | 0 | 0 |
Primer 2 (10 uM) | 1 | 8.2 | 8.2 | 8.2 | 8.2 | 8.2 | 8.2 |
Template Oligo (100 pM) | 2 | 16.4 | 16.4 | 16.4 | 16.4 | 16.4 | 16.4 |
Kapa 2X Master Mix | 25 | 205 | 205 | 205 | 205 | 205 | 205 |
nfH2O | 21 | 172.2 | 172.2 | 172.2 | 172.2 | 172.2 | 172.2 |
Total | 50 | 410 | 410 | 410 | 410 | 410 | 410 |