Sam’s Notebook: qPCR – Geoduck gonad cDNA with vitellogenin primers

Earlier today I made some cDNA from geoduck gonad RNA for use in this qPCR to test out the vitellogenin primers I designed on 20181129

I also used geoduck gDNA (162ng/uL; from 20170105) as a potential positive control, or as confirmation that these primers will not amplify gDNA.

Primers:

(SR IDs)[https://ift.tt/2G7zOSt

  • 1712
  • 1711

All qPCR reactions were run in duplicate. See qPCR Report (Results section below) for plate layout, cycling params, etc.

qPCR Master Mix calcs (Google Sheet):

Sam’s Notebook: Reverse Transcription – Geoduck gonad RNA pool

To be able to actually test the vitellegenin primers I made, I needed some geoduck cDNA.

I pooled 1uL of each of the following 12 geoduck gonad RNA (isolated previously from histology blocks):

  • 02
  • 04
  • 07
  • 09
  • 38
  • 41
  • 46
  • 51
  • 65
  • 67
  • 68

The concentration of the pooled sample was 95ng/uL.

Used 950ng of the pooled RNA in the following reverse transcription reaction:

  • 10uL RNA
  • 1uL oligo dT primers (Promega)
  • 4uL H2O
  • Incubate 10mins @ 70oC in MJ PTC-200 (no heated lid); immediately on ice after incubation.
  • 1.25uL of 10mM dNTPs (Promega)
  • 5uL 5x MMLV Buffer (Promega)
  • 1uL M-MLV reverse transcriptase (Promega)
  • 3.75uL HH2O
  • Final volume = 25uL
  • Incubate 42oC in MJ PTC-200 (heated lid); 5mins @ 95oC

Will run qPCR and then stored in Sam’s cDNA Box 2 in -20oC.

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