IGEM:MIT/2005/Monday, June 6th

MIT iGEM 2005 Laboratory "bootcamp" (run by Natalie Kuldell and Kate Bacon Schneider)

I. Tour of 68-074

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.