Team:Brown/Notebook/July12

From 2010.igem.org

Monday, July 12 2010

11:50 AM – placed cells in incubator (37°C)

  • 4 tubes of BL21 in 10 µg/mL tet media (LB)
  • 4 tubes of BL21 in 12.5 µg/mL tet media (LB)

Each 4 tubes have colonies from the four plates plated on 7/9:

  1. Bl21 is in tet-supplemented tet plates with pPTPi
  2. BL21 in tet-supplemented tet plates with pPTPi (lower concentration tetracycline)
  3. BL21 in tet-supplemented tet plate with RFP
  4. Bl21 in amp plate with RFP

L. lactis left in 30°C incubator grew after 63 hours. MRS broth has glucose (ordered June 23).

Placed L. lactis without pPTPi plasmid put into 30°C incubator at 12:40 PM.

Transformation of LovTAP and hybrid promoter

  • Thawed four XL1Blue aliquots and two BL-21 aliquots
  • Added 10 µl dH2O to well L8 on plate 2 and also 10 ul dH2O to well 20F plate 3.
  • Added 5 µl from well 20F to 2 Xl-1Blue aliquots
  • Added 1 µl from well 8L to one Xl-1Blue and one BL-21 aliquot.
  • Incubated aliquots on ice for 25 minutes.
  • Heat shock for 2 minutes at 42°C.
  • Ice for 2 minutes and addition of 500 µl media
  • Incubator at 37°C for 1 hour.
  • Spun at 6.5 k RPM for 3.5 minutes to pellet
  • Resuspended in 100 µl LB
  • Prepared IPTG plates with kanamycin
  • Plated out:
    • 1 plate RFP BL21
    • 1 plate RFP XL1B
    • 2 plates XLIB pLAC/mnt
    • 2 plates BL21 pLAC/mnt
    • 1 plate XL1B no plasmid
    • 1 plate BL21 no plasmid

(all on amp+ plates at standard amp concentration)

Plated out 5 plates of XL1B LovTAP (but low concentration of rehydrated DNA)

  • 1 plate null XL1B
  • 1 plate null BL21
  • 1 plate XL1B RFP
  • 1 plate BL21 RFP

Placed in 37°C incubator at 2:25 AM

Also requested more LovTAP from MIT, since our supply has been depleted and our transformations might fail.

  • Removed from incubator 4 plates with the pLAC/Mnt hybrid promoter (2 in Bl21s, 2 in XL1Bs).
  • Took 4 culture tubes, filled each with 5 mL LB and 50 µl ampicilin at 800 µg/ml
  • Took a colony from each plate except the one that exhibited no growth and two from another and placed them into the culture tubes. Placed culture tubes in the incubator to create an overnight culture
  • Also made one control (no colony picked).
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