Daniel:Notebook/PosSequencing/2017-7-10: Difference between revisions
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>Djacobse |
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'''Results''' showed nothing. I'll have to do qPCR to see if there was anything there to begin with. | '''Results''' showed nothing. I'll have to do qPCR to see if there was anything there to begin with. | ||
<li> | <li>qPCR</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Dilute 2 uL each 10 uM 2-Biotin-Uracil and Primer2 into 16 uL nfH<sub>2</sub>O (1:10)</li> | <li>Dilute 2 uL each 10 uM 2-Biotin-Uracil and Primer2 into 16 uL nfH<sub>2</sub>O (1:10)</li> | ||
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[[Image:PlateLayout20170710-emPCR8-benchmarks.png|350x250px]] | [[Image:PlateLayout20170710-emPCR8-benchmarks.png|350x250px]] | ||
<li>Run the single primer protocol (main difference is 45C melting temp</li> for ~40 cycles</li> | |||
</ol></ol> | |||
==qPCR Results== | ==qPCR Results== | ||
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So there was plenty of material ''after emulsion'' but it was mostly lost during the PNK/lambda reactions | So there was plenty of material ''after emulsion'' but it was mostly lost during the PNK/lambda reactions | ||
[[Category:PosSeq]] [[Category:20170706]] |
Latest revision as of 17:43, 11 July 2017
emPCR8 (Started Thursday July 6)[edit]
Dye Hybridization and Imaging[edit]
Since Saturday's results showed nothing, I'm going to try again but with the protocol the same as the first Lambda exonuclease test
- Dye hybridization
- Mix 20 uL ssDNA (lambda digested) and 20 uL 2X SSC
- Add 1 uL appropriate (10 uM) dye, A488-dc6 for sample 1 and Cy5-dc6 for sample 3
- Incubate in the dark (RT) for 15 minutes
- Magnet pulldown, remove supernatant and add 40 uL 2X SSC buffer
- Add 15 uL to a slide for analysis
- Seal coverslip with nail polish and wait approximately 30 minutes to dry
- qPCR
- Dilute 2 uL each 10 uM 2-Biotin-Uracil and Primer2 into 16 uL nfH2O (1:10)
- Prepare the following 7.2X (1X) master mix
- 14.4 uL 1 uM primers (2, 1 each primer)
- 115.2 (16) uL nfH2O
- 144 uL 2X Kapa SYBR master mix
- Aliquot 38 uL master mix into each lane
- Add 2 uL appropriate sample according to plate layout File:PlateLayout20170710-emPCR8-benchmarks.png
- Run the single primer protocol (main difference is 45C melting temp for ~40 cycles
Results showed nothing. I'll have to do qPCR to see if there was anything there to begin with.
qPCR Results[edit]
<gallery perrow=2 heights=250px widths=350px mode=packed-hover> File:PlateLayout20170710-emPCR8-benchmarks.png|Plate layout File:EmPCR8-20170710-benchmarks-plateCTs-normed.png|Plate CTs File:EmPCR8-20170710-benchmarks-rawcurves.png|Raw curves </gallery
So there was plenty of material after emulsion but it was mostly lost during the PNK/lambda reactions