WK11 + 12 - Easy Peasy DNA PDF

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Questions and answers concerning DNA PCR cycle....

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WK11 + 12 – EASY PEASY DNA: Isolation of total genomic DNA from peas

WHAT PROPERTY OF DNA POLYMERASE USED IN PCR IS ESSENTIAL? = heat stability. WHAT WOULD BE SUITABLE TEMPERATURE FOR A PCR CYCLE? = 95, 60, 72. WHY IS NEUTRASE TM ADDED TO THE EXTRACTION BUFFER DURING DNA EXTRACTION PROCESS? = to digest the protein to small peptide fragments. WHAT IS THE PURPOSE OF 60 DEGREES? = allows the primers to bind to anneal the two separated single strands of the target DNA sequence. WHY IS STAINZYME TM ADDED TO THE EXTRACTION BUFFER AT THE DNA EXTRACTION PROCESS? = to digest the plant starch that could co-precipitate with DNA. WHY DOES DNA PRECIPITATE AT THE INTERFACE BETWEEN THE OVER LAYED ETHANOL AND THE LOWER AQUEOUS LAYER? = ethanol is a less polar solvent suppressing the ionisation of DNA. Na+ and DNA = soluble, if suppress the ionisation then (NaDNA).

A260:A280 RATIO: Less than 1.8 – more protein than DNA

HIGH H2O

Over 1.8 – more DNA than protein 1.75 – 1.85 – pure DNA

WRINKLED PEAS = less starch, more H20 = higher osmotic pressure. MUTATION in SBE1 gene in rr wrinkled due to 800bp transposon element (jump genomes and insert randomly), doesn’t code the active form of SBE1 enzyme so can’t convert amylose to amylopectin. WHEAT is a hexaploid.

PCR EQUIPMENT:   

Single stranded DNA template X2 primers (provide OH- for polymerase). dNTPs

During separation of DNA using agarose gel electrophoresis, the DNA molecules migrate towards the (+) electrode as they possess a sugar phosphate backbone that is (-) charged. Q1) During the separation of PCR products, what components of the original PCR mix allows you to monitor how far the gel has migrated, so that you can be sure that your DNA has not moved out of the gel and into the running buffer around the (+) electrode. == inert red dye. Q2) Which region of the electromagnetic spectrum is used to visualise the Sybersafe DNA stain which intercalates into the ds DNA that you visualise in your gel? = = Visible blue...