Team:University of Chicago/Notebook/Damonwang

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Friday 2008-07-18

pUC8 CVX 336C/JM109 cultures: one is turbid and one is clear. Caulobacter plates are ready, will inoculate a starter culture. E. coli strain DW01 (pUC8 CVX 336C/JM109) frozen in 200uL 80% glycerol per 800uL culture (~16% glycerol final concentration), in Box A, slots 1 and 2. Caulobacter in 5mL and 50mL PYE, picked separately, incubated ~30C, 100rpm. Some medium dripped down the neck of the flask, but I flamed the area.

Saturday 2008-07-19

Couldn't get into the building to check temperature or OD600. Apparently Daniel Choi happened to run into someone leaving, and got in that way. The professor says to see Julie Caldwell about 24hr access.

Monday 2008-07-21

Julie Caldwell sent in a request to add me to the 24hr access list.

To do

  • check OD600 on Caulobacter cultures; if ready, freeze glycerol stocks.
  • Test E. coli glycerol stock DW01 (JM109/pUC8 CVX 336C) by streaking an LB-chloramphenicol plate
  • Pour PYE-Agar plates, 5 w/ and 5 w/o 2ug/mL chloramphenicol (CM). Have PYE plates, need to pour LB-CM plates. In fact, just make a liter each of LB and PYE.
  • Need to set up for making electrocompetent cells. Sterilize
    • 250mL flask
    • 2L flask
    • 3 4x250mL centrifuge bottles
    • 2L water (MilliQ)
    • 10% glycerol (in water? yes)
    • 2x30mL Corex tubes
    • 35x1.5mL Eppendorf

50mL Caulobacter culture is OD600=2.0, so overgrown. (Cecil, blanked with PYE)

New plan

  • Seed 50mL Caulobacter culture for electrocompetent stocks
  • Streak PYE plate from overgrown Caulobacter culture
  • Nora will make PYE
  • Pour LB-CM for streaking DW01 from glycerol

Tomorrow, Nora will dilute the 50mL culture and I will make electrocompetent stock. When the Caulobacter plate is ready, seed 50mL for glycerol stock

Nora's SOB media and my stuff for electrocompetent Caulobacter autoclaved (left autoclave, 15min fluid cycle (cycle 4?)---Schonbaum says with several liters of fluid, better to use the 30min fluid cycle).

All the glassware for electrocompetent Caulobacter autoclaved (right autoclave, cycle 2) and put on lower shelf under micropipetter tips.

Remind Jim and Soren that every label should have date and owner's name. Nora's media flasks just said "SOB".

Julie Caldwell sent in a request for my 24hr access.

Plated Caulobacter PYE and Caulobacter LB+CM. Oops.

Poured 6 LB+CM, now in 4C.

Should have autoclaved PYE in 250mL flask. Now just took a guess, since no sterile graduated cylinders available. Must wait for PYE to cool in cool water bath.

Caulobacter DW02 ~175rpm, 30C, PYE 50-75mL

E. coli DW01 streaked on LB-CM from glycerol.

Tuesday 22 July 2008

Time OD600
9:45 .006
11:52 .017
13;50 .033

Will come in ~11pm to dilute Caulobacter to appropriate concentration for Nora tomorrow.

16:00 Starter culure from streaked DW01 plate from glycerol at 37C, 250rpm, 5mL LB+8.75uL 2mg/mL CM

17:00 Starter culture from Crosson's DW01 plate picked to 30C, ~170rpm, 5mL PYE

The plate streaked 21 July 2008 from vastly overgrown culture has solid paths of colony---not Caulobacter because that's too fast

22:50 Our keycards still don't work, but apparently someone came out just as Nora was trying to enter.

DW02-21Jul was ~.5 OD600 at 22:00. Discarded all but 4mL---returned 2 to flask w. 9x >= 7.5mL PYE, added 400uL 80% glycerol to each of the remaining mL and froze -80C in A3 and A4

Wednesday 23 July 2008

Time OD600 Comment
10:00 1.43 Dilute just before 11:00
10:45 .14 Diluted ~700mL in 2L
13:40 .39

Made competent Caulobacter, DW02C in -80C A5-33 50uL/tube in 10% glycerol

Miniprep'ed 22Jul-DW01/DWP1 Tube #1 is ~27uL 87.6ng/uL in 4C DNA #1 Tube #2 is ~27uL 117.4ng/ul in 4C DNA#2

Nanodrop:

  1. Clean w. dH2O and Kimwipe
  2. Open ND-1000 software and choose Nucleic Acid
  3. Initialize with 2uL MilliQ
  4. Blank with solvent (e.g., elution buffer for a column miniprep)
  5. Between samples, wipe with dH2O on Kimwipe
Tube Concentration (ng/ul) OD260/OD280 OD260/OD230
1 87.6 1.90 2.37
2 117.4 1.92 2.29

Discarded 22Jul-DW02 since 21Jul-DW02 worked out after all.

Test-transformed DW02C with 1uL of each DNA (DWP1/Alfred) tube, incubated 2.5hours @ 30C, 200rpm, 1mL PYE, then spread 87ng-undil., 87ng-10xdil., 117ng-undil., 117ng-100xdil. at 30C, 23Jul08-22:00

Aliquot'ed from 21Jul PYE >8mL to 12mL culture tube in blue rack.

To do

  • Autoclave 2 MilliQ bottles
  • Sequence DWP1---Ask Harper
  • Make 10 PYE-CM and 6 PYE plates (Resolve to keep 10 of ea. on hand. Also 2L PYE)
  • Make 1L PYE (plus PYE for agar)
  • Transform G/R/C/Y-FP plasmids into E. coli, make glycerol stocks

Waiting

  • For NTA to make M15 Medium
  • For colonies on 23Jul-DW02C/DWP1-PYE-Agar to inoculate liquid culture

Friday 25 July 2008

262CFU on 87ng-10xdil., so ~301CFU/ng

To Do

  • Pick a starter culture into PYE-CM
  • Autoclave agar and pour plates (10 PYE-CM, 6 PYE)
  • Set up for SDS-PAGE with 8M urea
    • (HS kids made 1L of PYE-agar in one big flask! Aliquot into 5x200mL 4x200mL and 1x170mL in 500mL flasks and re-autoclave.)
  • Get nylon mesh and Buchner funnel
  • Email Marcia re: keys (Got kicked out of 216, computers moved to 210 with files intact, but no internet)
  • Figure out how to do phase contrast microscopy with oil immersion >60x objective

5mL PYE in centrifuge RM shaker, "40cm stroke"?, 175rpm, 30C, 25Jul08-16:00 from 87ng-10xdil

Autoclaved 370mL agar and 50mL PYE, left-autoclave cycle 4

Oops. Going to add CM now.

20000ug/mL * V = 5mL * 2ug/mL so V = really small

Make 200ug/mL stock (100x dil. off 20mg/mL stock), then add 5uL added 5uL, then 45uL more.

Spilled 4 of my PYE plates, so only 2 of them (for 6 total). Poured 8 PYE-CM plates successfully (200mL PYE agar + 20uL 20mg/mL CM)

Monday 28 July 2008

Tuesday 29 July 2008

Thursday 31 July 2008

Appendixes

Naming System

  • Cultures are named by starting date, species, medium, and a number. Only as many of these as will give a unique name, are used.
  • Strains are numbered sequentially, prefixed by DW to indicate that it's mine, all mine!
    • Maybe they should have a species field too. Except I'm not likely to have more than three or four strains ever.

Freezer Box Inventory

"Synthetic Biology Box A", in the top shelf of -80C freezer
Slot Date frozen Strain Description
1 18 July 2008 DW01 Crosson's E. coli JM109 carrying pUC8 CVX 336C
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