BL4840

1
BL4840
(Molecular Biology techniques)

• Lab 3
• 9/14 9/15

2
Lab3

• 1. PCR
• 2. Purify DNA with MOBIO ultra clean kit
• 3. Ligation into the pET TOPO vectors.
• 4. Electroporation and plating on LB agar
  Kanamycin plates
• 1 DNA labeling
• 2. DNA estimation with Nanodrop

3
Some questions you might have

• PCR more information will be given in 11th week
• No need to use ligase for pET TOPO vectors
• Klenow fragment (a derivative of E. coli DNA
  polymerase I) is used to incorporate a labeled
  dNTP in a template-directed polymerization
  reaction (3 fill-in reaction) on annealed
  oligonucleotides or natural DNA. (from
  http//www.piercenet.com/Proteomics/browse.cfm?fld
  ID39047FFD-092E-4836-AD26-A99FAE51D9B2FormatPri
  nt). Notice we didnt do PCR in probe labeling
• Handout about southern hybridization if you have
  trouble to find the legend in Molecular Biology,
  Weaver RF, here is reference for you (Modified to
  the kit we will use) (a) biotinylate probe DNA
  (which we will do tomorrow) (b) denature the
  probe. (c)hybridize (d)mix with avidin-alkaline
  phosphatase (we will use a different detection
  system. So now we add Streptavidin-Horseradish
  Peroxidase Conjugated). Avidin binds tightly and
  specifically to biotin in probe DNA. The function
  of Horseradish Peroxidase is for detection.(e) in
  stead of adding phosphorylated substrate as in
  the textbook, we will add Luminol and hydrogen
  peroxide(H2O2). (f) as the other slide shows,
  After luminol is oxidized by Horseradish
  Peroxidase in the presence of H2O2, the
  hybridized DNA is detected. Note HRP
  Horseradish Peroxidase the picture in the
  handout has been modified to the kit reagent (d)
  steapMix with avidin-HRP (the box is the photo
  copy of the paper slot) and (e) mix with
  Luminol H2O2, but in the picture, there are
  still phosphorylated compound was added, and in
  (f), phosphate group is removed by alkaline
  phosphase enzyme. Dont get confused by that.