Lab 11 summary

Summary of Lab 11 for BIOL 112 (Winter 2011). Week of Mar 21 2011.

DNA electrophoresis.

Genetics: Double recombination and mapping problems.

1DNA electrophoresis¶

• To separate DNA fragments based on their size
• Electrophoresing them through agarose gel
• In this lab: Genomic DNA, plus DNA fragment containing the fruit fly gene cdk cloned into a plasmid
  • Genomic DNA more complex than the cloned cdk gene
• Procedure:
  • Comb template placed in tray
  • Agarose gel heated, cooled, poured into tray; solidifies; comb template removed
  • To load DNA samples, use pipetter, try to place it inside well (kind of tricky)
  • DNA samples contain bromophenol blue - blue dye, to monitor progress of electrophoresis
  • Once the samples have been loaded, close, connect to power supply for a while
• What is cloning genes:
  • Purifying a particular gene away from the rest of the genomic DNA, putting it in a bacterium
  • To make the bacterium accept the carrier gene, we put it in carrier DNA called plasmids
  • We also give it antibiotics to give the bacteria a reason to accept the plasmids lol
  • To do this, we cut the plasmid open with a restriction enzyme
  • Restrictions enzyme cut any DNA at a specific sequence, usually 6 base pairs, called a restriction site
  • Then we cut the genomic DNA with the same enzyme, so hopefully the two end pieces stick together, mixing genomic and plasmid DNA
  • Then, put the plasmid+cdk DNA back into bacteria (process: transformation)
  • We throw in an an antibiotic, and only the bacteria that have accepted the plasmid DNA will grow (for the most part)
  • To make sure we only get bacteria with the DNA we want
  • One problem - how do we make sure we have the right DNA attached to the plasmid etc
    • Look at its restriction map using a restriction digest
    • Cut the gene with restriction enzymes, see if it has the correct restriction sites in correct places etc
    • Whatever
• Large fragments of DNA migrate more slowly than smaller fragments (so less distance travelled)
• ADD MORE STUFF LATER

2Double recombination¶

• If you have three pairs of genes, all on the same chromosome
• Double recombination should occur less frequently than single recombination (but less frequently than expected, due to interference)
• First, determine the order of the genes - abc or bac or bca
• Then identify the double recombinants - the least frequent class of progeny
  • Use that to figure out which gene is in the middle
  • Distance between middle gene and one outer gene: frequency of just that outer gene switching (%)
  • Same for other outer gene
  • Distance between outer genes: add the two distances above