Starting Anew

 Reamplification of Individual Fragments for Future Overlap PCR

October 2nd, 2022

Introduction

As discussed in last week's blogs, the three individual fragments (left, kanamycin resistance, and right) are going to be reamplified from D. radiodurans DNA and E. coli plasmid DNA to eventually make a successful assembly using Overlap PCR. This is important because over the past few attempts at Overlap PCR, no assembly bands were visible other than a smear. A couple photos of past gel electrophoresis of Overlap PCR products from the Summer are shown below. 

After early discussion with fellow classmates and Dr. Tuohy, it was hypothesized that failed Overlap PCR procedures were due mainly to the PCR conditions used; however, it is now hypothesized that the failed assembly attempts were also due to the kanamycin resistance middle fragment being amplified from E. coli plasmid DNA using the wrong primers, primers that contain different homologous regions that do not match those of the left and right fragments. This is a plausible cause because originally the kanamycin resistance fragment DNA was taken from other mentors who passed down the project to me; I did not amplify the kanamycin resistance fragment myself. The objective of this past week was to inoculate D. radiodurans in TGY broth to extract DNA and proceed with the left and right fragments amplification through traditional PCR, but since the broth culture showed little to no growth, it was placed back into the shaking incubator to grow over the weekend. Meanwhile, E. coli plasmid DNA was obtained from a fellow classmate for the kanamycin resistance fragment amplification. 

Procedure

Nanodrop reading of E. coli plasmid (named pREM) acquired from fellow classmate:

ng/ul = 5.9

A260/A280 = 1.95

A260/A230 = 2.84

The amplification of the kanamycin resistance fragment will use primers (namely primer 3 and 4) that contain homologous regions to the left and right fragments of the fbaA gene obtained from D. radiodurans. 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

To insure sufficient products, three identical amplification reactions are carried out. The PCR tubes are labeled Kan^r 1, Kan^r 2, and Kan^r 3. In each tube, the following is added for a total volume of 20 ul:

16 ul High Fidelity master mix

2 ul plamid template DNA

1 ul primer 3 (forward)

1 ul primer 4 (reverse)

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

Initial D: 95 *C, 3:00 min

D: 95 *C, 0:30 sec

Annealing: 61 *C, 0:30 sec (the average Tm of primers 3 and 4)

E: 72 *C, 1:00 min

Final E: 72 *C, 5:00 min

Hold: 4 *C, infinity

The reaction is continued in the Thermocycler for 1.5 hours. 

Results

The Nanodrop OD260 concentrations of the three Kan^r fragments after PCR are:

Kan^r 1: 692.3 ng/ul          A260/A280: 1.80           A260/A230: 0.57

Kan^r 2: 669.1 ng/ul          A260/A280: 1.80           A260/A230: 0.56

Kan^r 3: 692.8 ng/ul          A260/A280: 1.81           A260/A230: 0.57

Agarose Gel Electrophoresis of the PCR products is carried out using a 2% gel (0.6 g agarose + 30 mL TAE buffer). Since the DNA concentration of the three fragments is high, only 5 ul of DNA were loaded on the gel along with 2 ul UView loading dye and 5 ul PCR water. The gel is run at 85 V for about an hour. The results are shown down below. The order of the three identical fragments is Kan^r 1 in well 3, Kan^r 2 in well 5, and Kan^r 3 in well 7. A 100 bp ladder is loaded in well 1.

As seen, all three fragments of kanamycin resistance are viable and of the correct band size (~ 947 bp including overhang). In the upcoming week, the other two fragments, left and right, will be amplified after the extraction of D. radiodurans' DNA from the growing broth culture.