Each Lab Pair Isolates Genomic DNA

1
Each Lab Pair Isolates Genomic DNA

• Thymus (Lorraine Schepis)
• Wheat Germ (Irene Wu)
• Spinach (Nicole Brasof)
• Rose (Carol Champion)

2
You will use common chemicals instead of lab
solutions to isolate genomic DNA

• Buffered aspirin as the buffer
• Palmolive or Wisk instead of SDS
• Isopropanol instead of lab alcohol (ethanol)
• Sterile saline as the diluent
• Adolphs meat tenderizer or contact lens cleaner
  instead of phenol or protease to destroy nucleases

3
Common Steps in Different Isolation Procedures

• Dissolve (wheat germ) or disrupt tissue
• Add detergent to disrupt cell membranes and also
  to help degrade cell walls
• Add meat tenderizer or contact lens cleaner to
  degrade nucleases
• Overlay lysate with alcohol to precipitate the
  DNA
• Collect the precipitated DNA by spooling or
  centrifugation
• Dissolve DNA in saline or TE

4
Digesting Your Genomic DNA with EcoRI

• Each lab pair should choose best genomic sample.
• Each pair will prepare three different digests of
  this genomic DNA sample, varying the amount of
  DNA, as described on p. 36.
• Incubate the digests until 130 pm.
• During this time, prepare an agarose gel.
• Also, prepare 2 samples of undigested genomic DNA
  as described on p. 36.

5
Pour An Agarose Gel

• Set up gel device, inserting a black dam at each
  end of the gel bed.
• Seal the joints with agarose.
• Pour your gel as you did yesterday.
• Overlay gel with TAE buffer after gel solidifies,
  as time allows.
• Pull comb as you did yesterday.

6
Prepare Samples From Your Digests

• Prepare 3 tubes by adding 15 µl TE and 2 µl dye
  and labeling tubes 1, 2, and 3.
• After the incubation of your 3 genomic digests
  ends, put digests at 65o C for 5 min. (Why?)
• Cool digests in ice.
• PREPARE A SAMPLE FROM EACH DIGEST by transferring
  5 µl from each digest to the prepared tube with
  the same number.

7
Gel Electrophoresis of Genomic Digests

• Finish preparing your gel if not already done.
• Load 20 µl of each of 3 samples (not the digests)
  as directed on p. 37.
• Begin electrophoresis as you did yesterday.
• Stain and photograph gels in ethidium bromide to
  increase sensitivity. Follow directions in
  manual.

8
Preparation of Bacterial Strains

• One person of each pair should streak one LB-Amp
  plate with JM101/pUC19 and another LB-Amp plate
  with JM101/pRAS2 as described on p. 38.
• This partner will use these plates as the source
  of bacteria for their plasmid DNA isolation
  tomorrow.