pGLO MUTAGENESIS
SESSION TWO
Transformation by Electroporation
You have already tried the chemical method of transformation; i.e., the use of CaCl2 and the heat shock. A more recent method is what is called electroporation. Electroporation has been shown to produce many more transformants than even the best chemical methods of transformation. In this method, to make cells competent, the culture is grown overnight and centrifuged and washed several times with cold water and finally with a glycerol solution. These competent cells and the plasmid mixture are placed between two electrodes and are subjected to high voltage. The high voltage creates temporary holes in cell membranes, through which macromolecules such as DNA can pass. Once the electrical field is removed, the pores reseal and the transferred DNA molecule becomes part of the cell.
With electroporation, it is important to have a salt-free preparation to get the best results. If salts are present in the solution, there will be a spark that usually kills all or most of the cells and the time constant (the time that voltage was applied to your sample in seconds) would show a value of about 1.0. It has been our experience that good preparations would have a time constant value of between 2.2 to 8.0. One other factor in poor preparations is that the "Actual Voltage" delivered to the cells would be much lower than that set by the experimenter. It is prudent to check the "Act Volts" button at the end of the run and make sure that the actual voltage delivered was equal or close to the "Set Volts".
Transformation efficiency as measured by the number of transformants per µg of DNA is usually 109-1010 depending on the voltage, length of pulse and the distance between the electrodes. There is usually a survival rate of 30-40%.
Laboratory Supplies
Eppendorf tube, 0.5 ml1/group
pGLO DNA, 10 ng/µl1 µl/group
Electroporator1/lab
Electroporator cuvette, 1 mm gap1/group
Electrocompetent cells (XL-1 Blue)50 µl/group
SOC broth in bottle1/lab
Microcapillary pipet tips1 box/table
Pipetmans, P20, P2001/group
Pipetman, P10001/lab
Pipet tips, large and small1 box of each/group
Pipetman holder1/group
L plate4/group
L+Amp plate4/group
L+Amp+Ara plate4/group
L+Kan4/group
L+Kan+Ara4/group
L+Amp+Kan2/group
L+Amp+Kan+Ara2/group
Spreader and alcohol beaker1/group
Procedures
The students at a table are divided into two groups, one on each side.
Competent Cells
1.Each group places a sterile electroporator cuvette on ice. The TA distributes a vial of competent cells (XL-1 Blue Electrocompetent Cells from Stratagene) to each table. Keep this vial on ice all the time. This vial contains at least 80 µl of electrocompetent E. coli cells (AmpS, KanS, GFP-) on ice. Each group transfers 40 µl of XL1-Blue cells to their ice-cold electroporator cuvette using a microcapillary pipet tip, making sure the cells are delivered to the very bottom of the cuvette slot. One side marks their cuvettes “Tn” for transposition. The other side marks their cuvette “C” for control. The two groups need to decide which would be the Tn and which the C group. Cuvettes are kept on ice.
Transformation by Electroporation
- The side with the “Tn” cuvette does the following:
-Use a microcapillary pipet tip to add 1.0 µl of transposome mixture to the cells in the cuvette (higher amounts could bring in too much salt into the solution).
-Change the pipet tip and set the Pipetman at 50 µl and mix the contents of the cuvette by drawing the mixture in and out a few times. Keep the cuvette on ice until electroporation.
- The side with the “C” cuvette does the following:
-Use a microcapillary pipet tip to add 1.0 µl of 10 ng/µl of pGLO to the cells in the cuvette and mix the plasmid and cells with a fresh pipet tip.
3.Carry your ice bucket with your cuvettes to the electroporator on the center bench. Set the voltage to 1,800 volts on the electroporator by pressing UP or DOWN arrows. (Generally 12,500 volts are used per cm cuvette gap).
4.The SOC broth (room temperature) and a P-1000 Pipetman are located next to the electroporator. Measure 1 ml of SOC broth into the Pipetman but do not add it to the cuvette yet, just have it ready to be added immediately after electroporation.
- Take your cuvette out of the ice, remove its lid (keep the lid in a sterile condition) and rapidly wipe its sides of ice and excess moisture with a Kimwipe and place in the machine (there is only one way that the cuvette would fit into the slot). Push the holder and cuvette into the machine. Press the PULSE twice and wait until the machine beeps and stops.
6.Rapidly open the holder and add the SOC broth to the cuvette. If SOC is not added immediately, most of the cells would die. Take the cuvette out of the holder. Mix the contents of the cuvette several times using the same pipet tip. Replace the lid.
7.Press on the "Time Const" button on the machine and record the time that voltage was applied to your sample in seconds.
Note 1: If the Time Const value was between 4.0 and 6.0, your sample has been salt-free and you should expect to get high transformation efficiency. On the other hand, if the value is around 1.0 or there is a crackling sound, the salt concentration has been rather high in your sample and you will have a lower number of colonies appearing on the plates.
Note 2: The actual voltage (ACT VOLTS) that the machine applied to your sample should not be far from 1800.
- Incubate the cuvette at 37°C for 60 minutes. Meanwhile, the “Tn” group should obtain and label two plates of each of L, L+Amp, L+Amp+Ara, L+Kan, L+Kan+Ara, L+Amp+Kan and L+Amp+Kan+Ara. The C group will need two plates each of L, L+Amp, L+Amp+Ara and L+Kan
Note: Label plates before moving them from the center table to your work area. Since all the plates look alike, labeling at the instance of removing them from the sleeves diminishes the chances of mistakes.
9.After cell incubation is complete, use an alcohol-flamed spreader to distribute 10 µl and 50 µl aliquots from your cuvette on each set of plate types. Do not move plates for 15 minutes to allow the liquids be completely taken up by the agar. Invert the plates and incubate them at 37°C for 40-48 hours. Your TA will take your plates out of the incubator in due time and will store them in the refrigerator until the next session.
Use of any section of this Lab Manual without the written consent of Dr. Eby Bassiri, Dept. of Biology, University of Pennsylvania is strictly prohibited.
Results of pGLO Mutagenesis Lab Exercise
pM: Session Two
Name ______Date ______Section ____
Names of partner(s) ______
1.Before you look at the results on the plates, fill out the following table with what your expectations would be. In front of each plate type, write Growth or No Growth based on whether you expect to see growth or not? Add the word Fluor. if you also expect the growth to be fluorescent under long wave UV light. Write your reasoning for each of your choices under the table.
______
Description of growth
______
Plate type Control Tn
Amp Mutants GFP Mutants
______
L
L+Amp
L+Amp+Ara
L+Kan
L+Kan+Ara N/A
L+Amp+Kan N/A
L+Amp+Kan+Ara N/A
2. Our main objective is to isolate GFP and Amp mutants. How do you propose to do this for mutants of GFP alone, Amp alone and GFP and AMP together from the plate type containing Kan?
3. If you inoculate an L+Kan+Ara plate with a colony from an L+Kan plate, what do you expect to see? Explain.
4. If you inoculate an L+Amp plate with a colony from an L+Kan plate, what do you expect to see? Explain.
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