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Team:Brown/Notebook/July6
From 2010.igem.org
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:
- Re-ligate with purified, cut pNoTat + WillRS.
- 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.
