validate+cDNA+clone+work+2-1-13

refer to Start planning to validate cDNA clones 1-7-13

I do not know if it would be best to just synthesize 1st strand cDNA 1st, and then perform the PCRs, or if I should stick with my original plan and amplify the specific genes I want straight from the RNA. I do not know if I have enough reagents to perform all of the reactions straight from the RNA.

In either case, I am out of 3' SMARTer CDS primer, and I should order some more primer that can amplify the poly DT region.

poly dT primer portal 2-1-13

How many PCR reactions do I want to do? refer to (Tumor Validation primers 1-28-13) RS61+FS61 RS61Tr+FS61 RW17+FW17 RW17Tr+FW17Tr RCX3+FCX3 RCX3Tr+FCX3Tr RRS8+FRS8 RRS8Tr+FRS8Tr REA+FEA REATr+FEATr RR130+FR130 RR130Tr+FR130Tr RR130WT+FR130 RH1+FH1 RH1Tr+FH1Tr RM1+FM1 RM1Tr+FM1Tr Rco1WT+Fco1 Rco1+Fco1 Fco1Tr+Rco1Tr

So that is 20 rxns.I think I should have enough reagents if I use the PrimeScript RT and the PrimeStar Max polymerase as I would like to do. I'll use 5-50 ng poly A mRNA. I'll do the reaction with the tumor cDNA as well as the normal cDNA.

These 20 rxns (T1-20 (T for Tumor) and L1-20 (L for liver) 2-7-13) were placed in Tumor Library 11-29-12 KW -20 C Box

For the sequencing I would like to reverse transcribe the whole gene from the 3' end. I'm not sure if I need to include the 5' cap end primer or not RS61 RW17 RCX3 RRS8 REA RR130 RR130WT RH1 RM1 Rco1

So that is 10 rxns

For the reverse transcriptase reaction I will need RNase Inhibitor

Needed more PCR Grade Water and Andrey told me I can get some from a large stock.

The images of the gel can be found here: S:\Research\Cancer_Eradication\Discovering tumor specific antigens\Tumor cDNA Library\2-7-13 tumor cDNA pcr

An annotated diagram of this experiment can be found here "C:\kurt\storage\CIM Research Folder\DR\2013\2-8-13\tumor clone validation 2-8-13\tumor clone validation 2-8-13.svg"

Summary document here "C:\kurt\storage\CIM Research Folder\DR\2013\2-12-13\Tumor Clone Validation Summary 2-8-13.docx"