Large Scale Experiment Design
From Genome@Yale
Designing for large scale experiments requires careful considerations and multiple verification steps prior to actual experiments.
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generating qPCR primer sets for a large number of targets
- ask Tae to demonstrate all the steps.
- obtain and format sequences as fasta or single sequence for each line (from UCSC genome browser).
- note: mask repeats and avoid 3' end of primers at potential SNPs.
- use makeinput.pl script (or derivatives) to format the sequences for inputing into Primer3
- use Primer3 to pick primer pairs
- using pcr.pl script (or their derivatives) to check/map PCR primers back to the genome
- this in silico PCR will also provide genomic coordinates for each PCR product and can be used to remap old primers designed for old build of the genome.
- do this at the end of day, if checking more than several hundred primers.
- need UserAgent perl module to compile the pcr.pl script
- if human genomic DNA is being amplified in mouse or other genomic contexts, check to make sure that primers don't amplify the other genomic sequence
- give primer list back to Tae for order
- when primers arrive, set up a small test set (two 8-well strips) to verify PCR conditions
- be economical and reduce PCR reaction volume when possible, so test if 5uL reactions would work with the templates
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designing qPCR primers for RT-PCR
- pick target sequence toward the 3' end of the mRNA, especially for long mRNAs
- choose RefSeq mRNA first, if no RefSeq is available then knownGene RNA, followed by AK#### annotated transcripts, then other RNAs.
- determine exon-exon junctions by examining the mRNA alignment to the genome.
- using a text editor find the splice junction and place brackets a couple of nucleotides at the junction.
- also mask potential SNPs
- use web version of primer3 or offline version depending how many genes are being tested.
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