Plasmid Transformation Lab Objective: I can specifically explain how to clone a gene of interest by inserting it into a plasmid via restriction enzymes and transforming bacteria.

Textbook Example: Step 1 Isolate “gene of interest” by cutting it out of human genome, using…what? (you just learned!) restriction enzymes Should only use one specific restriction enzyme that cuts just before and after gene (2 restriction sites)

Textbook Example: Step 2 Have bacteria with a plasmid that contains… 1) ONLY 1 restriction site that is the same as what was used to cut out gene of interest - INSIDE of a lacZ gene… 2) A gene to resist ampicillin: antiobiotic that kills bacteria Huh? lacZ gene? More in a bit…

Textbook Example: Step 3 Allow gene of interest inserted into plasmid Will note that not always successful: some plasmids seal up without insertion (nonrecombinant) Induce transformation: make new bacteria take up the plasmids

Textbook Example: Step 4 Grow on special plate Will filter out undesired: Bacteria that did not take up plasmid (did not transform) cannot grow in ampicillin plate Plasmids without gene of interest (no insertion) still breakdown lactose and appear differently

Distinguish the Experiments (still very similar) Textbook example shows restriction enzymes with transformation all in one experiment! The ONLY thing we’re doing is transformation (NO CUTTING WHATSOEVER) Objective: put plasmid w/gene into bacteria Company made a plasmid with said gene via restriction enzymes (they did cutting) We cause bacteria to take up that plasmid, which transforms it, since now has gene

pBLU Transformation Lab – What we’re doing It’s called pBLU, because that is the name of the plasmid being transformed Has 2 genes ampR Ampicillin resistance ᵝ-galactosidase Able to break downX-gal thanks to restriction enzymes Do you think EVERY plasmid in the source has both genes?

pBLU Transformation Lab – What we’re doing Background vocabulary: LB = Luria Broth (food for bacteria) Can be in liquid form in a tube Can be in agarose gel form in slant/plate AMP = Ampicillin (antiobiotic) Prevents bacteria from growing, UNLESS bacteria has a gene to break it down… X-gal = 5-bromo-4-chloro-3-indolyl-ᵝ-D-galactoside Substance that if broken down turns the cell blue (cell must have gene to do so)

pBLU Transformation Lab – What we’re doing Behind the Scenes: Background Making Different Types of Plates

pBLU Transformation Lab – What we’re doing Behind the scenes Background: How to isolate “competent” bacteria = 1) Desire bacteria that will transform (no foreign plasmids) 2) Desire bacteria that will grow quickly Keep refrigerated

Isolating competent bacteria Using sterile techniques! Get 1 colony (genetically identical) that is most competent: will transform and grow QUICKLY But wouldn’t the heat…? Only want THIS bacteria, so will need to… Why would isolation make cell grow more quickly?

Let’s go through procedure together! This is your last chance to look @ lab packet together! 2 tubes LABELED With plasmid (+) Without plasmid (-) Both get CaCl2 (sterile pipette) Buffer solution (250 uL) Sterilize/dispose pipette (bleach) Keep on ice (Moderates reaction) Add isolated colonies to each w/ sterile loop Suspend cells (mix/”dissolve”) w/pipette NO AGAR! (Sterilize/dispose loop: bleach)

Let’s go through procedure together! Add DNA plasmid only to + tube Dip in source and get a bubble in sterile inoculating loop Mix into + tube Sterilize/dispose loop: bleach “Incubate” both tubes on ice for 15 minutes – stabilizes plasmids and cells Have the DNA plasmids gone INTO the bacterial cells yet?

Let’s go through procedure together! Heat Shock step: 42°C for 90 seconds -why? To make bacteria cells take up plasmid Don’t forget to gently agitate (mix)

Let’s go through procedure together! Return to ice for 1 minute (stabilize) Add 250 uL LB to each tube (food) – sterile pipette Gently tap to mix Place tubes in rack for 5 to 15 minutes (recovery)

Let’s go through procedure together! Use sterile pipette to put 100 uL of cells in “correct plates” in a sterile way: “clam shell” method Is it okay to have drop clustered in one or several spots?

How to “sterile-ly” spread bacteria around Best choice Sterile Glass Beads (Alt.) Sterilized Spreader/Roller Requires alcohol/fire -may ruin

Status of the two tubes after transformation Bacteria with no plasmid? Yes Bacteria with plasmid? No Bacteria with no plasmid? Yes Bacteria with plasmid but no genes? Yes (not our fault) Bacteria with plasmid and genes? Yes (hopefully)

6 Types of Plates – SEVERAL CONTROLS! Will there be bacterial growth? How much growth? What kind of growth (just white or any blue?)?