validate cDNA clone work 2-1-13

2014-08-29

azim58 - 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

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

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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"

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