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Team:Brown/Notebook/July9
From 2010.igem.org
Friday, July 9 2010
Results of 7/8 overnight plates:
- Control RFP (ng amount) showed colonies, higher density than usual (because we were using Gary’s competent XL1-Blues)
- Diluted control RFP – showed no colonies
- pGEM transformation (with iGEM, and with Gary’s reagents) – both showed colonies, mostly blue spots indicating an unsuccessful ligation.
- 1 to 2 colonies were white – we picked them and place in LB culture.
- 1 colony from pGEM (Gary reagent) and pGEM (iGEM reagent) placed in 2.5 mL of LB with 2.5 µl of Amp 100 µg/mL; for total of 100ng/mL amp media.
- Placed at RT, 3:00 PM
- Placed at 37°C at 5:00 PM
Did miniprep of XL1-Blue/empty pNoTat, following Qiagen Kit protocol; eluted in 45 µl Buffer EB after letting sit for one minute to maximize DNA concentration.
Recommended Standard Antibiotic Concentrations:
- Ampicillin - 100 µg/mL
- Kanamycin – 25-50 µg/mL
- Tetracycline – 12.5 µg/mL
Transformation results
pPTPi – transformed BL21 plates grew lawns
We may try supplementing with more tetracycline. Current concentration is 5 µg/mL.
- Supplement: 1 µg/mL
- Stock: 12.5 mg/mL
Supplemented two plates with 50 µl of 10 µg/mL tetracycline.
Transformation of pPTPi into BL21
- Thawed BL21s (4 tubes, 150 µl cells each) on ice
- 2.5 µl of pPTPi (two tubes) and 1 µl of RFP control (2 tubes)
- (12:56 PM) – Incubate for 30 minutes on ice.
- Heat shock at 42°C for 1.5 minutes
- Incubate for 2 minutes on ice
- Add 100 µl LB, plated, left on for ~15 minutes and put into 37°C incubator at 2:00 PM.
Growing L. lactis using 30°C incubator in B8 MRS plates w/ 0.5% glucose.
