DNA Extraction and PCR of Individual Fragments

Deinococcus radiodurans DNA Extraction and Traditional PCR of Left and Right Fragments of fbaA 

October 16th, 2022

Introduction

Since the decision was previously made to start anew by re-extracting the individual fragments, two broth cultures of Deinococcus radiodurans were grown over the period of 4 days and measured for concentration. The OD600 readings of the two cultures were:

LBT (stands for Light Blue Tube): 1.51

DBT (stands for Dark Blue Tube): 1.60

Briefly, following protocol of the DNeasy UltraClean Microbial Kit, D. radiodurans DNA was extracted from both of the broth cultures above. This blog does not go into the details of the DNA extraction because there are previous blog posts from February 25th, 2022 and November 13th, 2021 that demonstrate the procedure of DNA extraction in detail. The ending volume of DNA in Elution Buffer is 50 ul, and the OD260 readings of both tubes are:

LBT DNA: 95.5 ng/ul                           A260/A280: 1.90                     A260/A230: 1.85

DBT DNA: 105.9 ng/ul                       A260/A280: 1.88                     A260/A230: 1.83

Performing a traditional PCR using the correct primers of the left and right fragments should result in high resolution single DNA bands in each well on an agarose gel. 

Procedure

Using LBT DNA as the template, the amplification of the left and right fragments of the fbaA gene will utilize primers 1 (forward) and 2 (reverse) for the left fragment and primers 5 (forward) and 6 (reverse) for the right fragment. 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 ensure sufficient products, six PCR reactions are carried out, 3 for the left fragment, and 3 for the right fragment. The PCR tubes of the left fragment are labeled L1, L2, L3 and of the right fragment R1, R2, R3. In each tube, the following are added for a total volume of 20 ul:

16 ul High Fidelity master mix

2 ul LBT 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 Nanodrop OD260 concentrations of the three left and three right fragments after PCR are:

L1: 560.1 ng/ul          A260/A280: 1.85           A260/A230: 0.57

L2: 557.2 ng/ul          A260/A280: 1.86           A260/A230: 0.56

L3: 576.0 ng/ul          A260/A280: 1.85           A260/A230: 0.57

R1: 561.3 ng/ul          A260/A280: 1.86           A260/A230: 0.57

R2: 570.4 ng/ul          A260/A280: 1.85           A260/A230: 0.57

R3: 584.9 ng/ul          A260/A280: 1.85           A260/A230: 0.58

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 left and three right fragments is high, only 3 ul of DNA were loaded on the gel along with 2 ul UView loading dye and 7 ul PCR water. The gel is run at 85 V for about an hour. The results are shown down below. The order of the fragments is:

L1 in well 1, L2 in well 2, and L3 in well 3, 100 bp ladder in well 4, R1 in well 5, R2 in well 6, and R3 in well 7. 

As seen, all three left fragments of fbaA are viable and of the correct band size (~ 582 bp including overhang), and all three right fragments are also of the correct size (~ 647 bp including overhang). In the upcoming week, Overlap Extension PCR will be re-attempted using the left and right fragments from this PCR procedure and the middle fragment from the previous PCR procedure.