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Team:University of Lethbridge/Notebook/GeneralLabAugst
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August 03, 2008
Nathan Phillips
Tranformation of pUC19:
1. Thaw E.coli DH5a cells on ice 2. Add pUC19 DNA, pipette gently to mix (1μl of plasmid) 3. Let sit for 30 minutes on ice 4. Incubate cells for 30 seconds at 42oC 5. Incubate cells on ice for 2 min 6. Add 1 mL SOC at room temp 7. Incubate for 1 hour at 37oC on shaker 8. Spread 100-300 μl onto a plate made with appropriate antibiotic 9. Grow overnight at 37 °C
August 5, 2008
Nathan Puhl, Alix, Roxanne and highschool students
Objective: Isolate 76bp band from non-specific amplified bands of riboswitch PCR
Using Qiagen MinElute Kit extracted two very, very similiarly sized bands after running on a 3.5% agarose gel.
Objective: Cut out LacI gene and open DT vector for construction
Restriction Digest of BBa_C0012 (LacI) and BBa_C0012 (Double Terminator)
LacI - EcoR I + Spe I
DT - EcoR I + Xba I
Overnight incubation at 37 C followed by 65 C for 15 min (enzyme deactivation)
Reaction Mixture:
-5 uL template -5 uL NEB 2 buffer -0.5 uL BSA -1 uL restriction enzyme #1 -1 uL restriction enzyme #2 -37.5 uL ddH2O
August 6, 2008
Nathan Puhl, Roxanne, Alix
-Ran a 1% Gel with the Lac I and DT restriction products.
-Performed a Gel Extraction of the restriction products using the Qiagen Gel Extraction Kit.
