Laboratory 1 Flashcards
What was the set up for practical 1?
(3)
BL21 E.Coli strain was grown and chemically treated with CaCl2 to render the BL21 permeable to plasmid DNA
Cells were then incubated with plasmid DNA and heat shocked at 42 degrees for the uptake of plasmid into BL21
Cells were cultured on a plate and then inoculated with LB broth and incubated again at 37 degrees
What should be done with all reagents prior to the practical?
(3)
They should be prepared in advance
They should be autoclaved and allowed to cool to 4 degrees
They should be kept ice cold
Describe what you did in the first part of the experiment
(5)
Pellet the cells from your culture and discard supernatant
Add 10 ml of ice cold 0.1M CaCl2 and resuspend
Pellet cells again and discard supernatant
Add 500ul of 0.1 M CaCl2
Incubate cells on ice for 15 mins, shaking occasionally
Describe how you carried out the transformation of competent E.Coli
(6)
Add 100ul of suspension into two eppendorfs, one with pGLO vector (+ control) and one blank (- control)
Incubate on ice for 20 mins, mix occasionally
Heat shock at 42 degrees for 2 minutes
Add 1 ml of SOC medium and incubate at 37 degrees for 15 minutes
Add 300 ul of transformed cells onto LB agar plates (LB agar, LB agar + ampicillin, LB agar + ampicillin + arabinose)
Incubate for a week
Which plate should fluorescence after a week?
The LB agar + ampicillin + arabinose
Stock arabinose = 250mg/ml
We want 100ug/ml arabinose
jononoobo
Stock ampicillin = 100mg/ml
We want 100mg/ml
vvoenfenf
How many agar plates do you have?
Agar only (no pGLO)
Agar + amp (no pGLO)
Agar + amp + arabinose (no pGLO)
Agar only (w/pGLO)
Agar + amp (w/pGLO)
Agar + amp + arabinose (w/pGLO)
After a week how does each plate appear?
Agar only (no pGLO) -> lawn of E.Coli
Agar + amp (no pGLO) -> nothing
Agar + amp + arabinose (no pGLO) -> nothing
Agar only (w/pGLO) -> lawn of E. Coli
Agar + amp (w/pGLO) -> clusters of ampicillin resistant E.Coli
Agar + amp + arabinose (w/pGLO) -> Glowing E.Coli clusters
