Gel Electrophoresis of PCR Products from E. coli transductants

A Summary of Agarose Gel Electrophoresis 

December 20th, 2021

Introduction

After having set up and completed polymerase chain reactions PCR on chosen colonies from E. coli transductants GCC104, GCC108, GCC201, and GCC210, the products of the PCR are now ready to be viewed with the bare eye by performing an Agarose Gel Electrophoresis. Gel electrophoresis is the procedure used to separate DNA bands based on their charge and size; DNA fragments are negatively charged, so by passing electrical current through the agarose gel, they move towards the positively charged end of the gel bridge in the electrophoretic chamber. In this experiment, the previously collected PCR products in the 16 PCR tubes along with a fluorescent dye are laid in two 8-well Bio-Rad horizontal gel electrophoresis systems to be later viewed with a UV Transilluminator for the presence and absence of the bands of interest. It is hypothesized that GCC104 and GCC108 will only show bands for the brnQ gene primers while GCC201 and GCC210 will show bands for the proY gene primers. This is because the first two are anticipated to be missing the proY gene and the second two to be missing the brnQ gene because of the transduction experiment which created quadruple mutants out of the E. coli strain CDC11-1. 

Materials and Procedure

To perform gel electrophoresis, the following materials are needed:

• Two 8-well Bio-Rad horizontal gel electrophoresis systems
• The gel - a solution of 1.5% agarose in Tris-borate-EDTA (TBE) buffer
• Bulldog Midori Green Xtra MG 10 dye
• TBE buffer
• 6X DNA loading dye 
• The 16 PCR products
• UV Transilluminator
• 1.5 mL microcentrifuge tubes
• Micropipettes 

To start, two gels mixtures need to be made by adding 0.45 grams of agarose to 30 milliliters of TBE buffer in two 125 milliliter flasks. Then the mixtures are heated in the microwave until the solution becomes clear and are given time to cool to 50 degrees Celsius. Subsequently, 2 microliters of the Bulldog Midori Green dye is added to both of the gel solutions in the flasks which will later allow the DNA bands to fluoresce under the transilluminator. After setting up the dams of the two electrophoretic system in place along with the 8-well combs, the molten gel mixtures are poured between the dams and allowed time to solidify. 

    Next, the 16 PCR products are assigned microcentrifuge tubes labeled 1 through 16, and 10 microliters of each PCR products are transferred to the parallel microcentrifuge tube. Each of the tubes receives 4 microliters of the DNA loading dye with a separate micropipette and the mixture is stirred with the pipette tip until uniform throughout. This step helps ensure that the DNA samples sink to the bottom of the wells made in the gels by increasing their density. A step of brief centrifugation follows to collect the liquid to the bottom of the tubes. 

    Now, the gels should have solidified, so the dams and the combs are removed from the electrophoretic system. The wells created by the combs should be on the Negative Pole of the systems. Afterwards, TBE buffer is added to the chambers on both sides of the gels until it completely covers the gels. The 16 PCR mixtures are then assigned to the two electrophoresis systems in the following manner (assume that the 8 wells of each system are added up to equal 16, and numbers 1-4 and 9-12 are assigned to the first system while 5-8 and 13-16 are assigned to the second system):

• GCC 104 samples go in wells 1-4 of the first gel
• GCC108 samples go in wells 5-8 of the second gel
• GCC201 samples go in wells 9-12 of the first gel
• GCC210 samples go in wells 13-16 of the second gel

With this is mind, the first gel electrophoresis system should have samples from GCC104 and GCC201 while the second system should have samples from GCC108 and GCC210. This mixed alignment ensures that the bands present would be identical in both gels (each gel has bands from proY and brnQ genes) when viewed under the transilluminator. After having transferred all of the samples (14 microliters total volume in each sample) to the above determined wells with separate micropipette tips, the lids of both systems are sealed to the top and both systems are connected to a power supply to start running electrical current through the gels for about 45 minutes. The two dyes in the gel should start to separate and move to the Positive Pole of the systems indicating the movement of the DNA bands. 

Results and Conclusion

The systems should be disconnected from the power supply when the dyes are within 1 centimeter of the opposite end of the gel. It is now time to view the gels under the UV Transilluminator to check for the presence and absence of the bands of interest. It is expected that wells 1-4 and 5-8 only show bands for the brnQ primers while wells 9-12 and 13-16 show bands for the proY primers. This is because it was hypothesized that GCC104 and GCC108 were proY::kan (negative for proY gene), but GCC201 and GCC210 were brnQ::kan (negative for brnQ gene). The actual results are shown in the picture below. 

As seen, the first gel has all the bands in the correct wells according to the transductant's DNA; however, the second gel has one of its bands in the incorrect well, suggesting that an error occurred during the experiment. To elaborate, GCC210 should have shown both bands for proY primers in wells 13 and 14 because it is expected to be missing the brnQ gene. Nonetheless, one of the bands is present in well 15 instead of 14, signaling that either some fragments of the brnQ gene still exists in the GCC210 transductant DNA or that a loading error occurred.

    Possible errors include loading errors with the PCR tubes which underwent the PCR reactions, errors with loading the correct PCR product in the correct microcentrifuge tube, errors loading the DNA loading dye in each tube, or errors loading the correct microcentrifuge tube content into the correct well on the gels. Controversially, a very unlikely error could be the case when the transduction was carried out. It is possible that the transduction procedure to create quadruple mutant GCC210 was not successful, meaning that some of the brnQ gene DNA is still present in the mutant, but this is very unlikely since only one band for brnQ showed in well 15 of the second gel. This information reinforces the claim that a human loading error occurred during the procedure, and this is not improbable since the procedure was carried out by multiple laboratory students under slightly varying conditions. The difference in conditions may include the timing of the loading, the amount loaded, and whether each student accurately followed every step of the procedure. In conclusion, this Agarose Gel Electrophoresis procedure will be repeated to confirm the results, and based on this the hypothesis shall be accepted or nullified. 

References

Deutch, C.E. (November 2021) Gel electrophoresis of PCR products. Put Project at GCC.