Reverse Transcription (Promega M-MLV: Cat#M1701; ) [Cost per sample ~$1.50]
A single reaction volume = 25uL. The volume of RNA, primer(s) and M-MLV RT used are variable and will be specific to your current experiment. The directions below apply to a reaction using 1ug of total RNA. You may need to make changes to accommodate your own conditions.
5 uL 5x Buffer (M-MLV RT Buffer)
1.25 uL 10mM dNTPs (Promega Cat#U1511)
0.5 uL M-MLV RT per ug of RNA
7. Mix well.
8. Add 6.75uL of master mix to each reaction.
9. Mix well, but do not vortex.
11.Incubate @ 42C for 1hr in thermalcycler for oligo dT primers OR @ 37C for random primers.
12.Heat inactivate @ 95C for 3 min.
14.Store @ -20C.
Starting RNA 1ug calculations
2-1 1.13 ul
2-2 1.50 ul
Results: Procedure was going well until we reached the Master Mix solution. noticed that their was to much master mix left over so it is likely somewhere in the creation of the MM or pipetting the MM into the solution that an error was made. We still ran the entire protocol to see if anything was successful. Planning on running the PCR next week to verify that the primers that were ordered are valid.
Checked on the Sea pens today the changes to the peristaltic pump appeared to be successful everything was running correctly and the levels in the feeding bucket were the same as when I left yesterday. I am still considering removing the reverse flow on the pump into the bucket so the quantities of artemia being delivered are more consistent but will watch the sea pens to see if this makes a difference. I noticed that in each of the buckets with two sea pens that 1 sea pen is very inflated and looks healthy and the other does not so it is possible that they do not do well when in close quarters with each other possible because they are attacking each other when touching. I would like to take some tissue samples to verify that they in fact do have nematocysts though the I will have to look into if it would be visible just by looking through the microscope may need to find a way to fire them off which we had done in lab I will talk with Claire to see.