Team:Brown/Notebook/July6

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Tuesday, July 6 2010

Results of yesterday’s transformation:

  • Null plate: everything grew (control RFP, pGEM, pNoTat).
  • Amp++ XL1-Blue transformation with RFP  grew normally, no contamination.
  • Amp++ Xl1-Blue / pNoTat  no growth
  • Amp++ Xl1-Blue / pGEM  no growth.

This suggests that there’s nothing wrong with our plates or bacteria… they just didn’t get the plasmid. Or, there’s something wrong with our plasmid.

2 options:

  1. Re-ligate with purified, cut pNoTat + WillRS.
  2. Restart from the double digest… purify, ligate, and follow through with transformation.


Created another liquid culture of E. coli XL1-Blues transformed with pWill1. 3 mL of LB + scraped colony from plates (XL1-blue with control amp) (XL1-blue 6/24/10 labeled tube)

pGEM – TA overhangs on WillRS pNoTat – need sticky ends on WillRS

Ligation of pWillRS and pGEM T Easy

  • 7.5 µl 2X Buffer (rapid)
  • 1.0 µl T4 DNA ligase
  • 0.5 µl vector (pGEM T Easy)
  • 1.5 µl insert (Will RS)
  • 4.5 µl dH2O

15 µl total volume


  • 7.5 µl 2X Buffer (rapid)
  • 1.0 µl T4 DNA ligase
  • 0.5 µl vector (pNoTat)
  • 1.5 µl insert (Will RS)
  • 4.5 µl dH2O

15 µl total volume

11:50 AM begin at room temp

 use 5 µl for a transformation

Transformation of XL1-Blues with pNoTat, pGEM, RFP Control

Cell Competence protocol

11:27 AM Added 1.5 mL LB to cell culture to increase volume of overnight culture.

11:40 AM Placed cells on ice, to PRIMO for procedure

12:06 PM Spun at 4°C, 5 minutes, 3,000 RPM

12:11 PM Resuspended in 1 mL CaCl2 per aliquot. Placed on ice for 15 min.

12:27 PM Spun cells at 4°C

12:36 PM Resuspended in 250 µl CaCl2 per tube. Transformations were conducted with 150 µl cells each tube.

4:56 PM Cells spun down 1 min @ 3k RPM, Then 3 min @ 3k RPM, Then 3 min @ 3k RPM

Removed 250 out of 500 µl supernatant per tube.

Plated 50 µl per plate

Remaining cells placed in refrigerator (at 4°C).

Placed in 37°C overnight, at 5:15 PM

Took 3 µl of empty pNoTat plasmid, transformed ~150 µl of CaCl2 competent cells made earlier today.

Plated out at 7:15 PM on amp++ plate

Pelleted, reduced cell suspension volume to 250 µl.