1. 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.

2. 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)

3. 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…

4. 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

5. 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

6. 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

7. 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?

8. 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)

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

10. 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

11. 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?

12. Let’s go through procedure together!
This is your last chance to 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)

13. 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?

14. 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)

15. 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)

16. 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?

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

18. 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)

19. 6 Types of Plates – SEVERAL CONTROLS!
Will there be bacterial growth?
How much growth?
What kind of growth (just white or any blue?)?