Flashcards in Exercise 9: Background for Transformation Deck (13):
what are plasmids?
small extra chromosomal elements that can exist in some bacteria
what is the bacterial genome made of?
bacterial chromosome plus any plasmids that the organism may contain.
what do plasmids carry?
genes for antibiotic resistance that they can confer to the bacteria
what is a high copy number plasmid?
it may have up to 300 copies of itself in any one bacterial cell
what is pGLO?
it contains an ampicillin resistance gene, which codes for the exoenzyme ampicillinase.
what happens when the exoenzymes are secreted?
the ampicillinase goes out into the environment and destroys the ampicillin in the media
what strain of E. coli do we use in the lab?
E.coli JM 83; contains no plasmids, makes no restriction enzymes which means whatever plasmid we succeed in inserting, will not be cut up by any restriction enzymes
what type of plasmid is used in the Biorad kit?
Arabinose which is used to turn "on" the glow portion of this plasmid
what type of plasmid is in the plasmid from Biorad
pGLO which contains ampicillin resistance cassette and a gene for a green fluorescence protein(GFP). once expressed, the GFP will fluoresce under UV light. if the bacteria has taken up the plasmid, the colonies should fluoresce green under our UV light.
what is the procedure for the Biorad pGLO transformation ?
1. label each eppendorf tube with a + and -
2.using sterile technique, place 250 microliters of CaCl2, to help make the bacteria component to take up DNA.
3. place tubes in ice.
4. flame loop, inoculate big glob of component bacteria into + and - tube only.
5. transfer one loopful of the pGLO plasmid solution to the + tube, close it and put back in ice. close - tube and put it back in ice ( no DNA).
6. incubate in ice for 10 mins. this allows the DNA to attach to the CaCl2 and cell walls of the bacteria, and will facilitate transformation.
7. label 4 agar plates.
8. heat shock for about 50 seconds in 42 degree C water bath.
9.Add 250 microliters of LB ( Luria broth, named after great scientist Salvador Luria) nutrient broth in each tube and mix the tubes by inverting them and incubate for 10mins at 37 degree C.
10. Mix the tubes, then remove 100 microliters of the tube contents to place into each of the four plates.
11. spread the solution using the hockey stick method
12. stack the plates upside down, tape them with masking tape and incubate for 2 days at 37 degree C.
Explain the function of the CaCl2 and the heat shocking.
A. CaCl2: to help make the bacteria more component to take up DNA
B. heat shocking: alters membrane fluidity creating pores. allows for plasmid DNA to enter the bacterial cell.
why did you get no growth on the control plate NA/AMP?
Amp is a beta lactase and destroys the bacteria.