Hey Kim, this site shows the kids everything! NY ‘09 use this for the prelab stuff!! Michael.Gregory/files/Bio%20100/Bio% 20100%20Laboratory/Bacterial%20Tran sformation/transformation.htm

AP Bio Lab #6B pGLO Bacterial Transformation

Aequorea victoria has a GFP gene= a gene that codes for Green Fluorescent Protein

has been added to the pGLO plasmid GFP = a gene that codes for Green Fluorescent Protein

Lets the Plasmid map you will be inserting: ori = origin of replication

araC = operon promoter site remember: this is where RNA polymerase binds to the DNA

Plasmid map: bla = gene that codes for B-lactamase… a protein that makes resistance to ampicillin (it breaks the ampicillin down!!)

MATERIALS LB/amp LB/amp/araLB + -

4 plates + 1 STARTER PLATE +pGLO LB/amp w/ plasmid +pGLO LB/amp/ara w/ plasmid -pGLO LB/amp w/o plasmid -pGLO LB w/o plasmid LB/amp LB/amp/araLB + -

1 STARTER PLATE E. coli on Luria Broth (LB) media

Know your pipettor!!

HERE WE GO… In order to induce transformation competence, bacterial cells are first treated with an ice-cold solution of calcium chloride. It is believed that the positive calcium chloride binds to the negatively charged DNA strands. It then binds to the cell membrane

Transferring Bacteria Organisms are transferred by using a sterile loop and reaching in from the side while keeping the plate covered as much as possible. This technique minimizes the risk of contamination from above.

Immerse a sterile loop into the bottle containing plasmid DNA. When the center of the loop is coated with a soap- like film, transfer it to the “+” DNA microtube. Use a new sterile loop to transfer a second loopful of plasmid DNA into the same (+ DNA) microtube.

The - DNA microtube will not receive any plasmid DNA.

4 plates +pGLO LB/amp + = plasmid added +pGLO LB/amp/ara + = plasmid added -pGLO LB/amp - = no plasmid -pGLO LB - = no plasmid

The Arabinose Operon araC araB These code for digestive enzymes that break down arabinose sugar arabinose sugar araC araB RNA polymerase araC araB RNA polymerase mRNA

The Expression of GFP araC GFP Our plasmid’s ara Operon has been genetically altered, the regulatory genes have been replaced w/ the GFP gene from the jelleyfish arabinose sugar araC RNA polymerase araC araB RNA polymerase mRNA GFP

Results