Biology Lab

IB SL Biology Lab

Molecular Biology:

Transformation and Electrophoresis

Christina Qi

2/16/07

Aim:

How can a plasmid be engineered to include a foreign piece of DNA and how does gel electrophoresis separate DNA molecules present in a mixture?

Hypothesis:

If the pGLO plasmid is inserted into competent Escherichia coli cells, then the transformed bacteria will be resistant to ampicillin and will glow green under UV light. If samples of DNA are cut using certain restriction enxymes and separated using gel electrophoresis, then the smaller the DNA fragment cut, the greater the distance it will travel in the gel.

Variables:

The control plates used in transformation are the LB and second LB/Amp plates marked with a “-“. The control for restriction digest was the ladder. The independent variables are the different cultures of the E. coli, and the +LB/Amp and +LB/Amp/ara plates form the experiment group. The dependent variable is whether the cells glow green under the UV light and whether they are resistant to ampicillin or not. The variables in restriction digest are the other 4 samples. The dependent variable is the length that the DNA fragments travel while the independent variable is the size of the DNA fragments.

Method for Controlling Variables:

The variables were kept controlled by using different inoculating loops to add and spread out each solution. This was done to prevent any solutions from mixing together. The same amount of pGLO plasmid solution was added by dipping the loop until a bubble was formed across the loop opening. The tubes were incubated for the same amount of time and heat shocked for the same amount of time. The same amounts of Luria broth and cell suspension were used to keep the number of individual colonies controlled.

Materials Used:

For Transformation:

2 culture tubes (one marked “+” and one marked “-“)

4 sterile transfer pipettes

1 incubator (37Ð'o C)

4 LB plates (LB, 2 LB/Amp, LB/AMP/ara)

4 sterile inoculating loops (plastic)

1 bottle of Calcium Chloride (on ice)

1 beaker with ice

1 bottle of Luria broth

1 hot bath (42Ð'o C)

1 culture of E. coli in log phase growth

1 vial of plasmid (on ice)

Pencil and paper to record data

1 UV light box

For Electrophoresis:

1 vial of Lambda DNA cut with HindIII

1 vial of Lambda DNA cut with EcoRI

1 vial of Lambda DNA cut with BamHI

1 vial of Lambda DNA uncut

1 micropippetter (20 Ојl)

Masking tape

1 gel bed

1 UV light box

1 bottle of Ethidium Bromide

1 bottle of agarose gel

1 practice loading gel

1 power supply

1 comb

1 Electrophoresis chamber

1 bottle of buffer

1 marking pen

1 staining tray

Pencil and paper to record data

Ruler

Method for Collecting Data:

1. Mark the sterile 15-ml tubes with “+” and “-“. Use sterile transfer pipette to add 250 μl ice-cold calcium chloride to the tube. Place the tubes on ice.

2. Use sterile plastic inoculating loop to transfer one or two large colonies of E. coli cells from Starter Plate into your tubes (+ and -).

• Be careful not to transfer any agar from plate along with cell mass.

• Immerse loop in calcium chloride solution and vigorously tap against wall of tube to dislodge bacterial cell mass. Hold tube up to the light to observe that the cell mass has fallen off the loop.

• Immediately suspend cells by repeatedly pipetting in and out with sterile transfer pipette. Be careful not to get the cell mass stuck in the pipette.

• Examine against light to confirm that no visible clumps of cells remain.

3. Return the 15 ml tubes (+ and -) to ice.

4. Use a sterile plastic inoculation loop to add one loopful of pGLO plasmid solution (concentration is 0.03 μg/μl) to the +pGLO Tube. 10 μl is measured when DNA solution forms “bubbles” across the loop opening. Immerse the loopful of pGLO plasmid solution directly into cell suspension, and swish loop to mix the DNA.

5. Return “+” tube to ice, and incubate both tubes (“+” and “-“) on ice for 10 minutes.

6. While tubes are incubating, label the 4 petri dishes (LB, 2 LB/Amp, and LB Amp/ara) with your initials, date, and a “-“ or “+”.

• The LB/Amp/ara, and one of the LB/Amp plates marked with a “+” are the experimental plates.

• The LB and second LB/Amp plates marked with a “-“ are the controls.

7. Following 10 minutes of incubation on ice, “heat shock” the bacterial