TITLE: 16S and GyrB bacterial amplification
AUTHORS: Robert Nichols

[DESCRIPTION]
This protocol is used for the amplification of the bacterial gyrB gene and the 16S gene for both PacBio Sequel II and Illumina MiSeq sequencing. This protocol is used in the paper titled Long-read Sequencing Increases the Accuracy and Specificity of the gyrB Phylogenetic Marker Gene. 

 

[BEFORE_START]
Before starting make sure to have isolated bacterial DNA and selected primers for amplification.  

[STEPS]
SECTION: Prepare DNA for Amplification
1. Thaw the isolated  DNA 

SECTION: Prepare DNA for Amplification
2. Measure DNA concentration on the Nanodrop

This requires only 1 µL of isolated  DNA. Concentration values typically range from 100 ng/μl to 400 ng/μl. In addition, the NanoDrop gives only an estimate of the total  DNA concentration. For a more accurate result, submit samples for quantification on a Bioanalyzer.

SECTION: Prepare DNA for Amplification
3. Create a 100 μl aliquot at 10 ng/μl concentration.


SECTION: Prepare DNA for Amplification
3.1. Figure out how much DNA to add by dividing 1000 by the average DNA concentration.

For example: if your average DNA concentration was 254 ng/μl you would take 1000/254 which equals 3.94. So we would use 3.94 µL for the aliquot.

SECTION: Prepare DNA for Amplification
3.2. Figure out how much nuclease-free water to add by subtracting the result of step 3.1 from 100.

So to continue the example it would be 100 - 3.94 which equals 96.06. So we would add 3.94 µL of original DNA to 96.06 µL  of nuclease-free water. This results in a 10 ng/μl Bacterial DNA solution.

SECTION: 16S and gyrB PacBio and Illumina Amplicon PCR Protocol from Nichols et al.
4. Prepare the following PCR mix. 

 

SECTION: 16S and gyrB PacBio and Illumina Amplicon PCR Protocol from Nichols et al.
7. Make sure the reagents are mixed in the PCR tubes by gently flicking and then quickly spinning in a mini centrifuge. 
SECTION: 16S and gyrB PacBio and Illumina Amplicon PCR Protocol from Nichols et al.
8. Run PCR 
SECTION: 16S and gyrB PacBio and Illumina Amplicon PCR Protocol from Nichols et al.
8.1. If amplifying 16S samples for MiSeq use these PCR settings.


 


SECTION: 16S and gyrB PacBio and Illumina Amplicon PCR Protocol from Nichols et al.
8.2. If amplifying 16S samples for PacBio use these PCR settings.


 

SECTION: 16S and gyrB PacBio and Illumina Amplicon PCR Protocol from Nichols et al.
8.3. If amplifying bacterial gyrB samples for MiSeq use these PCR settings.


 



SECTION: 16S and gyrB PacBio and Illumina Amplicon PCR Protocol from Nichols et al.
8.4. If amplifying bacterial gyrB samples for PacBio use these PCR settings.


 


SECTION: Check for Amplification
9. Create a 1x agarose gel 

This is made by combining 1 g of agarose and 100 mL of 1x TAE. Microwave this solution for 1 minute and 45 seconds. Pour into a mold with an appropriate comb. Add 10 µL of Gel Red dye (at 10,000x). Let this cool for 45 minutes to an hour. 

SECTION: Check for Amplification
10. Prep the amplicons for gel electrophoresis 

First, prep the dye master mix as follows:

 

Then add in all 20 µL of amplified product to a well containing 24 µL of the dye water mixture. 

SECTION: Check for Amplification
11. Run the gel to check for amplification 

Add in all of the amplicon sample + dye + water mixture the wells of the submerged gel. Run the gel at 80 volts for 1 hour.

Check the gel in a geldoc to see amplified bands.  
SECTION: Clean the amplicon samples with a gel clean up kit
12. Cut out the appropriate bands from the gel from step 9

It is easiest to use specialized pipette tips to punch out the appropriate bands. Also, this needs to be completed under UV light so be sure to wear propper PPE.       
SECTION: Clean the amplicon samples with a gel clean up kit
13. Use the QIAquick Gel Extraction Kit to clean up gel punch-outs 

Briefly, dissolve the gel punch-outs in the provided buffer at 50°C for 10 minutes. Then add the dissolved punch-out mixture to the provided columns.  Wash twice with the provided wash buffers and elute with either nuclease-free water or elution buffer (not provided).   
SECTION: Clean the amplicon samples with a gel clean up kit
14. Submit samples for sequencing 

MiSeq samples were run on a 250x250 Illumina Miseq 

PacBio samples were run on a PacBio Sequel II 
SECTION: 16S and gyrB PacBio and Illumina Amplicon PCR Protocol from Nichols et al.
5. Fill an adequate number of wells with  19 µL of master mix each

SECTION: 16S and gyrB PacBio and Illumina Amplicon PCR Protocol from Nichols et al.
6. Add 1 µL of 10 ng/μl DNA directly into the master mix of each appropriate well 

It helps to watch the DNA go into the master mix to ensure that the DNA has been added.