Lab 6 (transformation) Flashcards
pBS transformation protocol
1) Use 3 microfuge tubes one with plasmid “A” (amp r, and lac+), plasmid “B” (amp r, and lac-) water “C” (water control amp s, lac-) Place each tube in ice.
2) Add 100 ul of cells (E. Coli) using clean pipettes and swirl the tubes gently to mix.
3) Incubate in ice for 15 min without disturbing to stick Plasmids to cell membrane.
4) heat shock : place the tubes in 42C for 50 seconds. Return them to ice for 2 minutes.
5) Add 900 ul LB broth to each tube and incubate at room temp for 20 minutes to let cells “recover”
4) plate them out
5) incubate overnight at 37 C
LacZ gene
Is an enzyme called beta-galactosidase (beta- gal) that splits lactose into glucose and galactose.
pGLO transformation protocol
1) Prepare cells –> wash cells in CaCl2 –> the washing is to penetrate the DNA = it helps to suck up the DNA during heat shock.
+. Wild host sensitivity : (lac-) no enzyme to break the glicosidic linkage, (amp s) ampicillin sensitivity
2) mix host cells with naked DNA –> plasmid DNA are used pGLO + and pGLO- (no plasmid)
3) let mixture “rest” on ice –> for ice to stick to outside of cells
4) Heat shock –> 50 seconds in water bath at 42 C –> then return to ice for 2 min.
5) add LB (liquid broth), and let cells “recover”
6) plate them out
7) incubate overnight at 37 C
What is the sugar used to switch on pGLO (plasmid green fluorescent protein)?
Arabinose
The pGLO plasmid contains also a ____________
_________ special gene regulation system which can be used to control GFP expression in transformed cells.
Transformed cells will appear _________ on plates NOT containing arabinose
White
Transformed cells will appear _________ on plates containing arabinose
Flourescent
A ________ precipitate will form when beta-gal acts on X-Gal (second chemical added). Thus, a ________ colony indicates that cells are ________ while a white colony is __________
This is to detect if the ________ gene was taken up by the cells
Blue Blue Lac+ Lac- Lac+
