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 @ 70^oC 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 42^oC in MJ PTC-200 (heated lid); 5mins @ 95^oC

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