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Team:Brown/Notebook/July16
From 2010.igem.org
Friday, July 16 2010
Results of test for cell competence:
XL1B from 6/28: hopelessly contaminated on Amp+
XL1B from 6/24:
- w/ RFP control: ~40 colonies---1.4 E6
- w/ Empty pNoTat: ~342 colonies---3.4 E6
BL21 from 6/24:
- w/ RFP control: ~46 colonies---0.46 E6
- w/ Empty pNoTat: ~74 colonies---0.74 E6
CFU calculation:
(pg plasmid DNA / total rxn volume)*plated volume = (100pg/250µl)*250µl = 100 pg DNA plated
(Avg # colonies / pg DNA plated) * 106 = # transformed cells / µg = CFU
- Control culture exhibited no growth as expected (LB+amp, overnight at 37°C).
- Poured amp+ plates (100ng/mL stock) – 1 mL in 1L LB produced 40 plates.
- Made glycerol stock of part BBa_J06702 in both XL1B and BL21s. Also miniprepped BBa_J06702 from the same overnight culture using Qiagen Kit.
- Made overnight culture of Bba_K191003 (aka LovTAP), which came in today
- 5 mL LB
- 3.57 µl Kan at 35 mg/µl
- NO IPTG, so now it is a low-copy plasmid (pSB2K3)
Adrian made competent cell stock (XL1B) frozen for us. We Aliquoted into 2.5 boxes full of 150 µl tubes.
Test competency of cell stock:
10 µl RFP diluted (100 picograms DNA) + 150 µl competent cells
Followed Adrian-modified protocol for transformation; plated onto amp+ plate and a null plate as a control.
Test of tet resistance in L. lactis
- 5 µl L. lactis with plasmid (pPTPi) transferred to 5 mL MRS
- 5 µl L. lactis without plasmid (pPTPi) transferred to 5 mL MRS.
Both tubes had 5 µg/mL tetracycline.
Test of tet efficiency in BL21s:
- 5 µl BL21 in 10 µg/mL tet LB
- 5 µl BL21 in NO tet LB
