Amgen Lab Possibilities

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Amgen Lab Possibilities
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Recombinant Sequence

• Lab 1 Pipetting and Electrophoresis
• Lab 2 Restriction Digest Using restriction
  enzymes (Hind III and BamH I) cut the p-ARA and
  the p-KAN-R forming 4 fragments (2 in p-ARA and 2
  in p-KAN-R)

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• Lab 3 Ligation Combine p-ARA and p-KAN-R
  fragments and ligate (glue) them together to make
  a recombinant plasmid. Goal to get p-ARA-R
  Plasmid, (which is a ligated plasmid containing
  the DNA fragment with amp resistance and an rfp
  gene)
• Lab 4 Confirmation of Restriction digest and
  Ligation.

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• Lab 5 Transform E. coli with recombinant plasmid
  (p-ARA-R) and plate cultures on Petri Plates with
  various media (LB, LB/Amp or LB/Amp/Ara).
  Incubate (grow) overnight. Next day look for
  growth of red cells on LB/Amp/Ara plate.
• Lab 6 Pluck one red colony and grow overnight in
  LB/Amp/Ara (this will be used for the whole class
  in lab 7)

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• Lab 7 Column purification of rfp protein.
• Lab 8 This is a totally separate lab (does not
  use any bacteria, or plasmid). Goal to extract
  human DNA and copy (through the process of PCR)
  the Alu gene, run it through a gel, stain, and
  view a sample of each individual students DNA.
  Analyze to see which students are Heterozygotes
  vs. Homozygotes

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Non-Recombinant Sequence

• Lab 1 Pipetting and Electrophoresis
• Lab 2a We work with two epi tubes. Both contain
  p ARA-R. The a- tube is our negative control, no
  enzyme added. The A tube will be cut using
  restriction enzymes (Hind III and BamH I), the
  p-ARA-R is cut into two fragments. Goal To
  produce a large fragment with the amp-r resistant
  gene and a second smaller, the rfp gene.

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• Lab 3 Ligation N/A
• Lab 4a Confirming that our enzymes worked (the
  plasmids were cut). The A-tube is uncut and used
  as a control to compare to our cut plasmids.
• Using Gel Electrophoresis to view the restricted
  fragments and comparing the cut lane with A-. We
  see the different forms of plasmids in the uncut
  lane (supercoiled, nicked circle and multimers).

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• Lab 5a Transform E. coli with supercoiled
  plasmid (p-ARA-R) and plate cultures on Petri
  Plates with various media (LB, LB/Amp or
  LB/Amp/Ara). Incubate (grow) overnight. Next day
  look for growth of red cells on LB/Amp/Ara plate.
• Lab 6 Pluck one red colony and grow overnight in
  LB/Amp/Ara (this will be used for the whole class
  in lab 7)

9

• Lab 7 Column purification of rfp protein.
• Lab 8 This is a totally separate lab (does not
  use any bacteria, or plasmid). Goal to extract
  human DNA and copy (through the process of PCR)
  the Alu gene, run it through a gel, stain, and
  view a sample of each individual students DNA.
  Analyze to see which students are Heterozygotes
  vs. Homozygotes