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.
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):
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):
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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