Team:UChicago/Notebook

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Revision as of 18:32, 24 July 2014 by Dbgasaway (Talk | contribs)

You should make use of the calendar feature on the wiki and start a lab notebook. This may be looked at by the judges to see how your work progressed throughout the summer. It is a very useful organizational tool as well.

This needs to get split up into separate pages somehow

Contents

Week 1

Day 1: 6/16/2014

KY

Plated some DH5-alpha competent cells in preparation for gDNA extraction and future transformations

Day 2: 6/17/2014

EJ, RK

Made LB Agar for plates- 3 Liters

  • The solution mix was overheated and flowed over. Remember to heat for only 1 min to dissolve everything except agar. Agar will dissolve during autoclave
  • Annie has edited the protocol
  • Also remember to autoclave some stir bars so antibiotics can be added while mixing on stir plate afterwards.

KY, RK

  • Made LB again
    • 1 L
  • Replated DH5-alpha cells, they’re in the 37 degree heater
  • Inoculated DH5-alpha cells for gDNA prep tomorrow (possibly boil prep?), they’re in shaker.

AZ

  • Made 1L SOB for preparing competent cells, with Kevin helping to adjust pH:
    • Protocol: http://parts.igem.org/SOB
    • For 1L,
      1. 5 g yeast extract
      2. 20 g tryptone
      3. 0.584 g NaCl
      4. 0.186 g KCl
      5. 2.4 g MgSO4
  • Made CCMB80 buffer for preparing competent cells, with Rikki helping to adjust pH:
    • Protocol: http://parts.igem.org/Help:Protocols/Competent_Cells
    • CCMB80 was stored in the 4 degree cold room.

EJ, RK

  • 20 chloramphenicol plates made
  • 20 plain agar plates made
  • 19 Kanamycin plates made

AZ

  • Wrapped the plates in parafilm
  • Stored in cold room behind centrifuges - not sure if we can store them there. Someone ask Rust lab about 4C storage
  • KanR plates were not labelled with green. Realized after wrapping so labeled green on parafilm.

Things we’ve borrowed

  • Rust lab
  • Tryptone (~50 g)
  • Pipette tips (1 box of 1000 uL tips)
  • 1 bag of 14 mL plastic conical tubes
  • 1 box of 15 mL conicals
  • Reagents to make 1 L of SOB (~ 20 g tryptone, 2.4 g MgSO4, .186 g NaCl)
  • Reagents to make 1 L CCMB 80
  • 1 syringe and syringe filter
  • Aluminum foil
  • Weigh boats
  • Jennifer
    • 1 mL SOC
    • LB kanamycin (~15 mL)
    • Inoculating tubes

KY

Plans

  • Need to grow up plasmids from iGEM distro kit tomorrow once plates are ready
  • Need to inoculate cells overnight
  • Need to replate cells
  • Make electrocompetency buffer.
  • Other buffers: I’ll make a list/figure out how to do everything tonight.
  • do gDNA extraction prep, PCR everything, run gels
  • start restriction digests (if time permits/everything looks good)

Summary

  • Made plates with different antibiotics for future use
  • Replated and inoculated DH5-alpha cells for gDNA prep
  • Made buffers for competent cell preparation.

Day 3: 6/18/14

EJ KY

  • Made 50ml 0.5M EDTA solution, pH 8.0, filtered
  • Made 50 mL 10% SDS solution
  • Made 10 mL 1% NaCl solution

RK

  • Made, filtered 250mL 1M Tris-HCl solution, pH 7.98
  • Made, filtered 80mL 3M sodium acetate solution, pH 5.2
  • New document in Reagents folder: cross-checked reagents,equipment we need with Gordon/Searle catalog (buy stuff tomorrow)
  • Resuspended 6 vectors from iGEM: iGEM 2014 wellplate 4 well 17O accidentally resuspended. iGEM 2013 wellplate 1 well 3M might have too much H2O, too dilute, *hopefully during transformation)

EJ KY RK

  • Inoculated 3 tubes (see Kevin’s post below)

RK EJ

  • Resuspended primers
  • Vortexed primers
  • Primers have been put in “-20” fridge.

KY

  • I forgot that the shaker from Rust lab was being adjusted to a lower temperature overnight, so there wasn’t much bacteria this afternoon :/. (Note: This means you should check the schedule that’s taped to the shaker before you put stuff in it to make sure the temperature will be OK and not be stupid like me)
  • We’re re-inoculating 3 tubes for today: 2 for making electrocompetent cells and 1 for the gDNA prep which has be en postponed to tomorrow. We’ll also do all the PCRs tomorrow and start the digestions/cloning if they look good (else rerun PCRs under different conditions for any that don’t look good).
  • We’re also inoculating the RBS-containing plasmid today so we can get miniprep tomorrow.
  • We’ll inoculate the starter culture tubes for electrocompetent cells into 250 mL media overnight to grow for the day after tomorrow (? actually not sure: protocol says to do it at 20 degrees C but we can probably do it at 37 degrees C which means it would grow a lot faster)
  • Made SOC, transformed the following 6 vectors from the iGEM kit plates (with chloramp and amp negative controls of course):
    • RBS 34 plasmid (BBa_B0034): 2014 Kit Plate 4, Well 1N on amp backbone
    • RBS 32 plasmid (BBa_B0032): 2014 Kit Plate 2, Well 2J on chloramphenicol backbone; 2014 Kit Plate 4, Well 1J on amp backbone
    • RBS34+T7 promoter plasmid (BBa_K525998): 2013 Kit Plate 1, Well 3M on amp backbone
    • Generic promoter that we prefer to use (BBa_J23119) (labeled with a P): 2014 Kit Plate 3, Well 17O on chloramp backbone; 2014 Kit Plate 4, Well 17B on amp backbone

Things we borrowed

  • Rust lab
  • 9.30 g EDTA borrowed
  • 5 g SDS borrowed
  • ~30 g Trizma base
  • ~4-5 mL HCl for pH adjustment
  • 1 250 mL filter
  • ~12.3 g anhydrous sodium acetate
  • ~2 50mL syringes
  • ~2 syringe filters
  • ~36 g glucose
  • 5 amp plates
  • ~8 tubes of 100 uL competent cells
  • Enough glass beads (~5-6 on average per plate) to spread 8 plates


Things I will be borrowing tomorrow

  • 2 mL 1:1 phenol:chloroform
  • Need to remember to sterile filter CCMB80
  • Need to check if we have 6X loading dye
  • Need to check what bp ladder sizes we have
  • Check parafilm?

Summary

  • Made solutions and buffers to be used in later procedures
  • Re-inoculated three tubes, two for electrocompetent cells and one for the gDNA prep
  • Transformed general primer and RBS vectors from the iGEM plates
  • Readied primers for use later
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