1. Tucson High School Biotechnology Course Spring 2010

2. pcr (polymerase chain reaction) dsDNA (double stranded) ssDNA (single stranded) dsDNA (double stranded) What do we need to do this?

3. dsDNA (double stranded) ssDNA (single stranded) primer polymerase dNTPs (dATP, dGTP, dCTP, dTTP) MgCl 2

4. Still won’t work… dsDNA (double stranded) ssDNA (single stranded) 94ºC dsDNA (double stranded) 52ºC primer 72ºC polymerase primer taq polymerase Thermus aquaticus

5. What primers are we using? psbA-forward GTNGAYATHGAYGGNATHMGNGARCC psbA-reverse GGRAARTTRTGNGCRTTNCKYTCRTGCAT DNApol-reverse GGIAYYTGIGCIARRTTIGG g23-forward ATTTGIGGIGTTCAGCCIATGA DNApol-forward GAYACIYTIRYIYTITCIMG g23-reverse CGCGGTTGATTTCCAGCATGATTTC DEGENERATE PRIMERS N - A, C, G, T H - A, C, T Y – C, T M - A, C R - A, G

6. Why use degenerate bases?

7. virus Bob A A C T G C A G T A A virus Emma A A C T G C A G T A A virus Rick A A C T G C A G T A A virus Estelle A A C T G C A G T A A PRIMER T T G A C G T C A T T

8. Why use degenerate bases? virus Bob A A C T G C A G T A A virus Emma A C C T A C C G T A A virus Rick A G C C G C T G T C A virus Estelle A T C C A C C G T C A PRIMER T N G Y R G H C A M T

9. Today… 1 Master Mix in 3 tubes dNTPs Taq polymerase MgCl 2 water + psbA primer + DNApol primer + g23 primer + virus DNA + virus DNA + virus DNA

10. What do we have to be aware of? psbA primer DNApol primer g23 primer psbA MM DNApol MM g23 MM CONTAMINATION