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.