amplify specific transcripts from RNA 3-18-13

2013-12-01

azim58 - amplify specific transcripts from RNA 3-18-13

Plan
-Develop conditions to amplify specific transcripts from RNA to

determine if any of the final transcripts from my tumor library screening
(see 8-26-12 Plasmids for verification) exhibit anything odd when I try
to amplify the whole transcript from the 1st exon to the last exon. I
will compare the results from tumor and normal RNA.

-Starting samples will be poly A purified samples.

-I can refer to the 2-1-13, 2-6-13 and 2-7-13 protocols to see how I

first tried to amplify the transcripts. I can then try several different
conditions to amplify a selected transcript. I will choose the Wfdc17
transcript to amplify so I will use RW17+FW17 primers to perform this
optimization. I'll try a few starting RNA amounts, cycle numbers, and
annealing temperatures.

--In the previous experiment I used 5 ng poly A RNA. I used 30 cycles

with an annealing temperature of 55 C.

-In the new experiment I'll try 5, 50, and 100 ng poly A RNA. I'll try

25, 30, and 35 cycles. I'll try 50, 55, and 60 C annealing temperatures.
see listing all combinations of conditions in an experiment

-Samples to use
--6-24-10 Tumor RNA
--Normal RNA from 1-29-13 (BALBsc mammary gland RNA)

-Primers to use
--RW17+FW17 and RW17Tr and FW17Tr (see Tumor Validation primers 1-28-13)

-Kits to use: PrimeScript RT and PrimeStar Max

Expected size of final amplified transcript: 470 bp

===========================================================================
3-20-13 performed PCR with PrimeSTAR Max

Spreadsheet of samples found here:
"C:\kurt\storage\CIM Research Folder\DR\2013\3-20-13\PCR\samples
3-20-13.xlsx"