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