IGEM:MIT/2005/Pipetman use, working w/ bacteria, PCR

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II. Pipetman use

  • Background Information/Demonstration (asked students what they knew):
    • settings on different Pipetman
    • different sizes of tips used (blue for P1000, yellow or white for P20, P200)
    • first stop to pull up liquid/push through to second stop to release liquid
    • releasing tips with ejector button-->dispose of in SHARPS
    • change tips frequently!
  • Exercise (each student):
    • pipette minimum and maximum from each Pipetman (1 µl and 20µl for P20; 20 µL and 200 µL for P200; 200 µL and 1000 µL for P1000) using Xylene Cyanol dye
    • allowed students to see how consistent pipetmen were, get a feel for what a certain volume looks like in an Eppendorf tubes

III. Sterile technique, serial dilutions, plating bacteria

  • Background Information/Demonstration (asked students to help explain):
    • keep sterile things sterile by not touching them with nonsterile things (fingers, benchtop, etc.)-->work CLEANLY!
    • flaming tubes after uncapping, after adding/removing liquid from tubes
    • dilutions: a 1:10 dilution is 1 part concentrated solution + 9 parts dilution (10 parts total)
    • serial dilutions:
      • dilutions "in series"-->make 1:10 dilution, take 1 volume of 1:10 into 9 parts diluent to create 1:100 dilution, etc.
      • More accuracy/less error than pipetting small volumes (e.g. 1 µL in 100 µL total)
    • plating cells:
      • label plates on media side with initials, date, strain name, dilution, etc.
      • add liquid to be plated to center of plate (keep lid face down on bench top while adding, then cover)
      • sterilize glass spreader by dipping in EtOH and passing through flame (don't bake spreader!)
      • touch spreader to plate away from cells (to avoid killing them!)
      • move spreader back and forth while spinning plate (about 45 seconds to "dryness"-->notice change in resistance as you spread)
  • Exercise:
    • Students made 1:10 and 1:100 serial dilutions of log phase bacteria and plated 50 µl of each (plus undiluted) on LB plates, then grew overnight at 37˚C

IV. Streaking bacteria for single colonies

  • Background Information/Demonstration:
    • the fabulous Beckwith lab 3 toothpick method for obtaining single colonies
      • remember: it's a serial dilution on a plate!
  • Exercise:
    • had students streak out 1 AmpR and 1 AmpS strain on an LB Amp plate, then grew plates overnight at 37˚C

V. Making media with antibiotic and inoculating liquid cultures

  • Background Information/Demonstration:
    • How to make LB Amp media from LB and Ampicillin stock solution (1000X working concentration)
    • how to inoculate O/N culture
      • use "stick" (dowel) and touch colony (gets ~106 cells on stick)
      • open tube (sterile technique) and touch stick to liquid media
      • flame tube and cap tube quickly
      • don't use small pick (have to drop toothpick in media, part of stick that touched your fingers is not sterile and will contaminate culture!)
  • Exercise:
    • had each group of 2 students make 25 mL of LB Amp media
    • each student inoculated 3, 3 mL cultures: one "media alone," one AmpR strain, one AmpS strain
    • grew cells O/N @ 37˚C

VI. Polymerase Chain Reaction (PCR)

  • Background Information:
    • Basics of DNA, DNA synthesis and PCR cycle
      • DNA
        • DNA is double stranded in E. coli cells
        • Two strands of DNA are held together by basepairing (A and T, G and C)
        • DNA has a 5' and 3' end (numbers= those of carbons of the sugar of the nucleotide...here deoxyribose)
        • DNA sequences written 5'->3' left to right
      • DNA synthesis
        • requires a primer (provides 3'OH for nucleotide addition)
        • proceeds in the 5'-->3' direction
      • PCR cycle:
        • Denaturation: separation of template strands (95˚C)
        • Annealing: primers bind via basepairing to template (50-65˚C, based on primer sequence and length)
        • Extension: DNA strand is "extended" from primers using the rules of basepairing (e.g. if template says A, add T onto primer) (72˚C, optimal temperature for thermostable polymerase)
    • Components of a PCR reaction
      • template (genomic DNA, plasmid)
      • primers (short DNA molecules, give specificity to amplification, usually ~1000X excess of template)
      • dNTPs
      • DNA polymerase (thermostable, like Taq, so don't have to add enzyme after each denaturation cycle)
      • pH buffer and salts
      • Mg+2 (required for DNA polymerase function, concentration can be altered in PCR reaction)
  • Exercise:
    • students set up PCR with template and primers from Natalie's lab
    • standard PCR master mix—ran cycle O/N.
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