Bacteria Transformation Flow Chart

Selena Felix Period 4

Question & Hypothesis

At this time you will rephrase the objective into a question.

Next, you will come up with a testable prediction on the outcome of this lab.

Materials
You will need the following materials for this lab: 2 micro tubes Sharpie marker Ice 2 cups Pipettes Sterline inoculating loop Hot water bath 3 plates The purpose of using ampicillin on the plates is to select for only those bacteria that took in the recombinant DNA plasmid.

Recombinant DNA Plasmid

ON or OFF
If a sugar arabanose is present then the red gene is ON.

Cells Solution
1) Using a sterile inoculating loop, several colonies of bacteria are picked from the top of the LB agar class starter plate.
Transfer the colonies to tubes of cold Ca++CI solution.

3)

Swirl and twist the loop/tip to make sure all the bacteria are mixed in the solution.
Place on ice.

2)

4)

Place the bacteria cells in a Ca++CI solution because calciumions help to neutralize the negative electrical charges of the plasma membrane and the plasmid.

Label Two Micro tubes

Tube 1
This tube will be Plasmid +

Tube 2
This tube will be Plasmid - (also known as the control)

Three Plates
Plate 1: LB (food) Plate2: LB / Amp (ampicillin) (antibiotic)

Make a line down the middle of plates 1 and 2. On one half place a P+ and on the other half place a P-.

Plate 3: LB / Amp / ara (arabinose sugar)

Mark a P+ on this plate

Micropipette
Set your micropipette at 050:
Add 50 uL of cells solution to both the + and tubes. (E.coli bacteria cells)

Micropipette
Using a second (smaller volume) micropipette set at 10.0:
Use a sterile pipette tips, add 10uL of rpARA plasmid to the positive (+) tube only.

Tap gently to mix.

Incubate
Incubate both + and tubes on ice for 10 minutes
We need to create a bit of pressure difference between the inside and outside of the bacteria cell. This is done by getting bacteria really cold and then quickly putting it in warm water, heat shock.

Heat Shock
Take your tubes in the ice water cup to the 42 degree Celsius hot water bath.
Make sure the water bath is at 42 degree Celsius. Transfer the tubes from ice to the hot water for 45-50 seconds heat shock. Immediately return the tubes to the ice for 2 minutes.

Membrane pores open in the heat so plasmids can enter

Room Temperature
Place + and tubes in a beaker/cup at room temperature.

Normalize bacteria cells:

Add 150 uL LB broth (food) to both tubes.

Mix / vortex gently.

Rest tubes for 2 minutes.

Plating Process
Use steril tip for loading. Use a new steril tip for + loading.

Tube:

50 uL onto Plates 1 and 2. Be sure to place on side.

+ Tube: 50 uL onto Plates 1 and 2. Be sure to place on + side. AND 50 uL onto Plate 3.

Steril Inoculation Loop

Using a steril inoculation loop, gently spread the sides of plates 1 and 2.

Using a steril inoculation loop, gently spread the + sides of plates 1 and 2, and then spread plate 3.

Incubate
Cover each plate and stack. After 15 minutes invert plates (agar side up). Tapes and label with team names

Incubate at 37 degrees for 48 hours.

Incubate at 37 degrees for 48 hours because it is a reproducing body temperature under ideal conditions.

My Prediction
Plate A: There will be A LOT of growth on both P+ and Psides.
Plate B: There will be some growth on the P+ side transformed and no growth on the P- side because there is no plasmid. Plate C: There will be a lot of growth transformed.

My Data
A: Control: Bacteria are viable able to reproduce.
B: Ampicillin: Ampicillin kills bacteria unless the resistance gene is on. C: Red: There is arabanose in the bacteria causing the gene to turn red. The bacteria transformed to red.

Ending Results
1. My actual results did not differ from what I predicted. I predicted that if a cell has the ampicillin resistance gene then the antibiotic ampicilin will not kill it.
2. The P+ bacteria growing on the LB / amp plate are like the P+ bacteria growing on the LB / amp / ara plate because they have a plasmid and ampicilin which is a resistance gene that is on. 3. The P+ bacteria growing on the LB / amp/ara plate are red because there is arabanose in it which turns on the red gene.