7. Agarose Gel Preparation Agarose : fine white powder; polysaccharide (galactose polymer) isolated from seaweed. 1% (w/v) dissolves in Tris-acetate buffer at ~60 ° C and the solution sets at ~30 ° C

8. Agarose Gel Image

9. Agilent Technology

10. Electropherogram showing Agilent analysis of total RNA 18S 28S Times (seconds) Fluorescence

14. HYBRIDISATION OVEN Incubate filter and probe in hybridisation buffer TREAT and BLOT GEL Transfer to nylon membrane nylon membrane and transferred DNA Southern/Northern Blotting and Hybridisation

18. Ligation of DNA Eco R I OH 3’ 5’ PO 4 T4 DNA ligase catalyses the formation of phosphodiester bonds PO 4 5’ 3’ OH T4 DNA Ligase T4 DNA Ligase Stu I Circular DNA Eco R I G CTTAA AATTC G

21. A General Laboratory Plasmid Multiple Cloning Site A foreign gene can be ligated into a plasmid, and the genetically engineered plasmid introduced into E. coli .

22. Cloning DNA into a Plasmid Both plasmid and foreign DNA have sticky Eco R I ends Insertion into E. coli (transformation) Agar plates contain antibiotic. Grow at 37 °C Place 1 colony in liquid media + antibiotic. Grow at 37 °C Purify Plasmid DNA (Billions of copies)

23. DNA and Retroviruses can serve as vehicles for the introduction of new DNA into a cell

24. DNA / RNA viruses as ‘vehicles’ Chromosomal DNA Viral DNA Integration into genome gene x Gene Therapy and Transgenics

27. A Thermocycler This thermocycler can accept 1500 reactions at a time, and complete them in 2 to 4 hours.

28. Principal of PCR A G C T A G C A T G T T G C G C G T A T C A T G T A C A G T G C A T A C G T C C C C T T A G C T | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | T C G A T C G T A C A A C G C G C A T A G T A C A T G T C A C G T A T G C A G G G G A A T C G A 5 ’ 3 ’ 5 ’ 3 ’ A G C T A G C A T G T T G C G C G T A T C A T G T A C A G T G C A T A C G T C C C C T T A G C T | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | 5 ’ 3 ’ | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | T o o l t o 5 5 ° C Cool. This allows specific ‘ primers’ to anneal as shown DNA (Double Stranded) Heat Denature (Becomes Single Stranded) Heat to 72 °C. Heat resistant DNA polymerase extends new DNA from the primers Heat to 72 °C C G A T C G T A C A A C G C G C A T A G T A C A T G T C A C G T A T G C A G G G G A A T C G A 3 ’ 5 ’ 3 ’ 5 ’ A G C T A G C A T G T T G C G C G T A T C A T G T A C A G T G C A T A C G T C C C C T T A G C T | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | G T A T G 5 ’ 3 ’ G T T G C | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | T C G A T C G T A C A A C G C G C A T A G T A C A T G T C A C G T A T G C A G G G G A A T C G A 3 ’ 5 ’ 3 ’ 5 ’ H e a t 9 5 ° C ( D e n a t u r e s ) A d d S p e c i f i c P r i m e r s C

30. Dideoxynucleotides ddNTPs have no 3’ OH, so when added they cannot form the phosphodiester bond required to add the next nucleotide

32. Electropherogram of sequencing gel

33. Decoding DNA sequence data

35. Primer Extension Theory SNP Analysis - primer extension theory

36. SNP Analysis