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 2.0 ul 5X First-Strand Buffer 0.25 ul DTT (100 mM) 1.0 ul dNTP Mix (10 mM ) 1.0 ul SMARTer V Oligonucleotide (12 ìM) 0.25 ul RNase Inhibitor 1.0 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