Daniel:Notebook/PosSequencing/2017-6-5: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Djacobse
>Djacobse
 
Line 47: Line 47:
File:20170605-aqPCR-EFraction-rawcurves.png|Raw curves
File:20170605-aqPCR-EFraction-rawcurves.png|Raw curves
</gallery>
</gallery>
[[Category:PosSeq]] [[Category:20170602]]

Latest revision as of 18:29, 9 June 2017

Aqueous PCR Test (Started Friday June 2)[edit]

Back to Calendar

Given the results from Saturday I'm going to repeat qPCR for the E fraction after baking for 15 minutes.

Protocol[edit]

  1. Baking
    1. Incubate fraction "E" for 15 minutes at 95C
    2. Chill on ice prior to adding to qPCR lanes
  2. qPCR
    1. Make the following 6.2X master mix (1X)
      1. 6.2 uL 10 uM BiotinUracilPrimer1 (1)
      2. 6.2 uL 10 uM Primer2 (1)
      3. 124 uL 2X Kap SYBR Master Mix (20)
      4. 99.2 uL nfH2O (16)
    2. Aliquot 38 uL master mix to appropriate lanes
    3. Add 2 uL sample according to plate layout below
    4. File:PlateLayout-20170605-aqPCR-EFraction.png
    5. Run the following thermocycler program
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x120
      7. 72C 2 min
      8. 16C hold

Results[edit]