GENSAT PCR design guideline

1. Log onto http://www.gensat.org/bacreport.jsp website. Locate the gene of interest then find the A-homology arm (A-box) 3' Primer for that gene.
2. Open another window or tab on your web browser and log onto http://www.ncbi.nlm.nih.gov/genome/seq/BlastGen/BlastGen.cgi?taxid=10090. In the search line type the name of the gene of interest and search the mapviewer by clicking on the Go button.
3. Under map element select a match that most closely fits your gene (this may need to be repeated if you do not see your gene on the chromosome map on the next page). If the gene is displayed, under maps and options click Download/View Sequence/Evidence. On the next page click display and highlight only the DNA sequence.
4. Open Vector NTI (or other sequence editor program), under file—click Create new sequence / Using Sequence Editor (DNA/RNA)…
5. Name the new DNA/RNA molecule then click on the Sequences and Maps tab and click edit sequence. In the new window click paste to paste the DNA sequence.
6. Using Find Sequence tab, enter the appropriate A-homology arm (A-box) 3' Primer (found in the BAC Report page) and search using both direct and complement. If it does not find the sequence, then reverse the sequence and search for that again by both direct and complement.
7. Mark the A-box primer sequence location by highlighting it or underlining it.
8. Locate the region of DNA transcriptionally upstream of the now marked 3’ A-box primer and highlight it.
9. Design a transcriptional sense gene specific forward primer (GSP) (this will be opposite strand of the 3’ A-box primer).
10. Analyze GSP candidates to ensure the Tm is between 55-60 and the GC content 40-60%. Pick primers low in secondary structure and with 3’ends (ΔG ~ -9kCal/mol) with 3 A/T and 2 G/C in last 5 nucleotides.
11. Use this new forward primer you just designed with one of the given eGFP reverse primer sequences (CAGGGCACGGGCAGCTT, TAGCGGCTGAAGCACTGCA, GGTCGGGGTAGCGGCTGAA, or CTTCGGGCATGGCGGACTT).
12. Use GENSAT Stringent Protocol first and then GENSAT Non-Stringent if needed.
13. Amplicon should yield >300 bp fragment on agarose gel (depending on placement of forward primer).
14. Optimize by: 1). Decreasing MgCl if nonspecific banding is prominent 2). Use final concentration of 1.3M Betaine and 1.3% DMSO in PCR cocktail if reaction is weak or fails.

Technician: David Hayes, 3/4/2009