Amplification of Potential Past 2-Way Assemblies

 Traditional PCR of Potentially Assembled Fragments

October 28th, 2022

Introduction

In hopes of running into a 2-way assembly product from all past Overlap PCRs, this procedure aims at amplifying the Left+Kanamycin and Right+Kanamycin products using traditional PCR. Over the past few months, multiple Overlap Extension PCRs were carried out either by the mentors Shawn and AJ or by myself after I took over the project. Since one Overlap Extension PCR was successful  in the past, this procedure aims to point to the assembly products that, hopefully, were not lost or used up in other procedures. It is hypothesized that at least one Left+Kanamycin product and one Right+Kanamycin product will show a favorable band on the gel. 

Procedure

Using old tubes labeled LM (left+middle) and RM(right+middle) as the template DNA because they were the assembly products of Overlap PCR, the amplification of the Left+Kanamycin fragments of the assembly products will utilize primers 1 (forward) and 4 (reverse). On the other hand, the amplification of the Right+Kanamycin fragments of the assembly products will utilize primers 3 (forward) and 6 (reverse). The concentration of the primers used is 5 micromolar. The PCR procedure utilizes the Q5 High Fidelity master mix with the following ingredients (17.5 ul volume of the master mix intended to be used per PCR reaction tube):

5X Q5 Reaction Buffer: 5 ul

10 mM dNTPs: 0.5 ul

Q5 High Fidelity DNA Polymerase: 0.25 ul

5X Q5 High GC Enhancer: 5 ul

PCR Water: 6.75 ul

For this PCR procedure, 7 past assembly products are being amplified, 3 of the Left+Kanamycin and 4 of the Right+Kanamycin. The PCR tubes of the Left+Kanamycin fragments are labeled LMU, LM7, LM8, and those of the Right+Kanamycin fragments are labeled RMU, RM7, RM8, and RMP. The "L" and "R" are for left and right, and the"M" in the naming denotes the middle (kanamycin resistance fragment), and the letter or number following that is the date of the product: "U" being unknown, "P" noting it was from a pink tube, 7 being July, and 8 being August. In each tube, the following are added for a total volume of 20 ul:

16 ul High Fidelity master mix

2 ul template DNA

1 ul forward primer 

1 ul reverse primer

The PCR conditions are as follows (* denotes degree symbol):

1) Initial D: 95 *C, 3:00 min

2) D: 95 *C, 0:30 sec

3) Annealing: 61 *C, 0:30 sec 

4) E: 72 *C, 1:00 min, GOTO Step 2 for 30 Cycles

5) Final E: 72 *C, 5:00 min

6) Hold: 4 *C, infinity

The reaction is continued in the Thermocycler for 1.5 hours. 

Results

The concentrations of the 7 PCR products were not taken after Traditional PCR; however, an Agarose Gel Electrophoresis was run to demonstrate the results. Agarose Gel Electrophoresis of the PCR products is carried out using a 2% gel (0.6 g agarose + 30 mL TAE buffer). In each well of the products, 5 ul of DNA were loaded along with 2 ul UView loading dye and 5 ul PCR water for a total of 12 ul. The gel is run at 75 V for about an hour. The results are shown down below. The order of the fragments is:

Ikb ladder in well 1, LMU in well 2, RMU in well 3, LM7 in well 4, RM7 in well 5, LM8 in well 6, RM8 in well 7, and RMP in well 8. 

As seen, there were no viable Left+Kanamycin or Right+Kanamycin products because no bands of the correct size (~1500 bp) showed on the gel. With this in mind, the hypothesis made above will be refuted, and all past Overlap PCR products will be discarded due to no visible assembly. In this upcoming week, Overlap Extension PCR will be re-attempted using a lower concentration of the primers (1 micromolar) and slightly different Tm conditions. Stay tuned for the next blog which will go over the new Overlap PCR procedure.