Start planning to validate cDNA clones 1-7-13

2015-01-13

azim58 - Start planning to validate cDNA clones 1-7-13


Plan for validating cDNA clones portal 1-7-13


I need to design the primers for the clones. I also need to design these
primers so that they can be cloned into the vector used with the
In-Fusion SMARTer Directional cDNA Library Construction Kit

I will amplify these genes from RNA to make 1st strand cDNA. Then I will
make 2nd strand cDNA. Then the genes will be cloned into the vector.

First
1–3.5 μl RNA (50 ng–1 μg total RNA or 50–100 ng poly A+ RNA)*
1 μl 3’ In-Fusion SMARTer CDS Primer (12 μM)
5’–CGGGGTACGATGAGACACCATTTTTTTTTTTTTTTTTTTTVN–3’ <-This will need
to contain the gene specific region rather than VN
x μl Deionized H2O

Then
The 1st strand reaction will use these components
  1. 0 ul 5X First-Strand Buffer
  2. 25 ul DTT (100 mM)
  3. 0 ul dNTP Mix (10 mM )
  4. 0 ul SMARTer V Oligonucleotide (12 ìM)
  5. 25 ul RNase Inhibitor
  6. 0 ul SMARTScribe™ Reverse Transcriptase (100 U)*

Next will be the ds cDNA synthesis

2 μl First-strand cDNA (from Step V.B.8 or V.C.8)
80 μl Deionized H2O
10 μl 10X Advantage 2 PCR Buffer
2 μl 50X dNTP Mix (10 mM)
2 μl 5’ PCR Primer II A (12 μM)
2 μl 3’ In-Fusion SMARTer PCR Primer (12 μM)
5’– CGGGGTACGATGAGACACCA-3’
2 μl 50X Advantage 2 Polymerase Mix

I will use PrimeStar rather than Advantage 2 at this step.

To design the primers for all of the genes simply pick a 5' primer from
the beginning of exon 1 that is in the same orientation as the original
gene.
Pick a 3' primer that is the reverse complement of the gene specific
region then add the normal kit 3' sequence to this primer.

see
"C:\kurt\storage\CIM Research Folder\DR\2013\1-7-13\validation of cDNA
clones\validation of cDNA clones 2 1-7-13.svg"
for a diagram
also here
F:\kurt\storage\CIM Research Folder\DR\2013\1-7-13\validation of cDNA
clones\validation of cDNA clones 2 1-7-13.png

Tumor Validation primers 1-28-13



===========================================================================