Setting Up PCR Reactions From Isolated Bacterial Genomic DNA

A Summary of PCR for E. coli Transductant DNA

November 29th, 2021

Introduction

Previously in the lab, genomic DNA from four colonies of transductant strains of CDC11-1 (GCC 104, 108, 201 and 210) was isolated and purified for performing polymerase chain reaction PCR. To recall, the two strains GCC 104 and GCC108 were suspected to be ProY::kan (proY negative) and the other two, GCC 201 and GCC 210, to be BrnQ::kan (brnQ negative). Also, the DNA concentration of each of the four strains was measured using a NanoDrop spectrophotometer. The average of the measurements was 222.2 nanogram/microliter for GCC104, 228.9 nanogram/microliter for GCC108, 224.8 nanogram/microliter for GCC201 and 65.4 nanogram/microliter for GCC210. Therefore, when preparing the PCR reaction for GCC210, three times the intended volume will be used because of its low OD reading. PCR reactions are aimed at amplifying pieces of DNA encoding a gene or genes of interest to determine the ways genomic DNA alterations can impact certain molecular functions inside species, given that this knowledge can lead to the advancement of the human life or the surrounding environment. 

Materials and Procedure

The following are needed to carry out PCR reactions for the E. coli transductant strains:

• Four sets of DNA primers (two for each of the proY::kan and brnQ::kan strains) designed and obtained formerly
• Four template purified DNA from the four chosen colonies GCC 104, 108, 201 and 210
• A 2X master mix containing nucleotides, a buffer, and magnesium ion
• A sample of Taq DNA polymerase
• Sterile water
• Two strips of 8 PCR tubes with caps
• Ice
• Micropipettes

Ahead of time, thaw out all of the frozen primers, template DNA solutions and the master mix on ice for about 10 minutes, and label the two strips of 8 PCR tubes with the numbers 1-16. 

First, 10 microliters of the 2X master mix needs to be transferred into each of the PCR tubes using the same micropipette tip. Then a volume of the template purified DNA is added according to its OD reading to each of the 16 tubes as such:

Out of the template DNA that has an OD reading of around 225 nanograms/microliter, add only 2 microliters to the corresponding PCR tube. That is, 2 microliters of the GCC104 is added to tubes 1-4, 2 microliters of GCC108 is added to tubes 5-8, and the same volume of GCC201 is added to tubes 9-12. On the other hand, since the OD of GCC210 was the lowest (about 65 nanograms/microliter), 6 microliters of this template DNA is added to tubes 13-16. 

Next, the primers originate from two types of genes, one is proY forward and reverse while the other is brnQ forward and reverse. For each type of primer, two sets are present in two different locations of the gene; the proY has primer sets 4 and 5, and the brnQ has primer sets 2 and 5. A microliter of the forward and reverse of each of the four primer sets is added to tubes 1-16 in such a manner that each of the template DNA in the tubes gets a forward and reverse of the proY primers 4 and 5 and the brnQ primers 2 and 5. 

Finally, the tubes are filled to 20 microliters by adding a volume of water in correspondence to the existing volume in the tube. Tubes 1-12 should now have 14 microliters of solutions, so 6 microliters of water are added to reach a volume of 20 microliters. However, tubes 13-16 need only 2 microliters of water due to the already existing volume of 18 microliters. 

For each of the tubes, a separate micropipette tip should be used to homogenize the solutions by drawing the liquid up and down. Once every solution has been mixed in each of the tubes, a step of brief centrifugation is required to collect the liquid from the walls of the tubes to the bottom. Afterwards, the two strips of PCR tubes are inserted into the heating block of the PCR device, and the parameter setup is as shown below. 

* Picture above is obtained from Dr. Deutch's handout

Conclusion

After the PCR reactions are complete, the PCR tubes are stored in the refrigerator for future use in Agarose Gel Electrophoresis. The PCR reaction should result in the amplification of the primers that exist in each of the GCC transductant strains. GCC 104 and GCC 108 are expected to experience the amplification of the brnQ primers 2 and 5 while GCC 201 and GCC 210 experience the amplification of the proY primers 4 and 5. That is because the first two transductants are missing the proY gene (ProY::kan) and the last two are missing the brnQ gene (BrnQ::kan). Hence, the amplification of certain DNA segments by PCR is an important step to physically visualize the genotypes of species that are genetically engineered through laboratory experiments like transduction, transformation, and others.

References

Deutch, C.E. (November 2021) Setting up PCR reactions. Put Project at GCC.