16S and GyrB bacterial amplification

Thaw the isolated DNA

Measure DNA concentration on the Nanodrop

This requires only 1µL of isolated DNA. Concentration values typically range from 100ng/μl to 400ng/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.

Create a 100 μl aliquot at 10ng/μl concentration.

Figure out how much DNA to add by dividing 1000 by the average DNA concentration.

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

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 3.94µL of original DNA to 96.06µL of nuclease-free water. This results in a 10ng/μl Bacterial DNA solution.

Prepare the following PCR mix.

A	B	C
Reagent	Concentration	Volume to make 20 uL of product
Forward primer	10 μM	0.4 μl
Reverse primer	10 μM	0.4 μl
Platinum SuperFi Master Mix	N/A	10 μl
Nuclease Free water	N/A	8.2 μl

This is for each well so multiply each volume by the number of samples plus one or two, to make sure enough master mix is available for all tubes. For example if I was running a 15 sample PCR I would multiply each volume product by 17 (15 samples + 2 extra)

Fill an adequate number of wells with 19µL of master mix each

Add 1µL of 10ng/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.

Make sure the reagents are mixed in the PCR tubes by gently flicking and then quickly spinning in a mini centrifuge.

Run PCR

If amplifying 16S samples for MiSeq use these PCR settings.

A	B	C	D
Cycle Number	Time	Temperature	Description
1 cycle	2 minutes	98°C	initial denaturation
25 cycles	10 seconds	98°C	denaturation
	20 seconds	56.6°C	annealing
	15 seconds	72°C	extension
1 cycle	5 minutes	72°C	final extension

It should be noted that the higher the number of cycles the greater chance for chimeric sequences. The user should optimize PCR cycles to their specifications

If amplifying 16S samples for PacBio use these PCR settings.

A	B	C	D
Cycle Number	Time	Temperature	Description
1 cycle	30 seconds	95°C	initial denaturation
25 cycles	30 seconds	95°C	denaturation
	30 seconds	57°C	annealing
	1 minute	72°C	extension
1 cycle	5 minutes	72°C	final extension

It should be noted that the higher the number of cycles the greater chance for chimeric sequences. The user should optimize PCR cycles to their specifications

If amplifying bacterial gyrB samples for MiSeq use these PCR settings. gyrB samples for MiSeq use these PCR settings.

A	B	C	D
Cycle Number	Time	Temperature	Description
1 cycle	2 minutes	98°C	initial denaturation
30 cycles	10 seconds	98°C	denaturation
	20 seconds	56.6°C	annealing
	15 seconds	72°C	extension
1 cycle	5 minutes	72°C	final extension

It should be noted that the higher the number of cycles the greater chance for chimeric sequences. The user should optimize PCR cycles to their specifications

If amplifying bacterial gyrB samples for PacBio use these PCR settings. gyrB samples for PacBio use these PCR settings.

A	B	C	D
Cycle Number	Time	Temperature	Description
1 cycle	30 seconds	95°C	initial denaturation
30 cycles	30 seconds	95°C	denaturation
	30 seconds	57°C	annealing
	1 minute	72°C	extension
1 cycle	5 minutes	72°C	final extension

It should be noted that the higher the number of cycles the greater chance for chimeric sequences. The user should optimize PCR cycles to their specifications

Create a 1x agarose gel

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

Prep the amplicons for gel electrophoresis

First, prep the dye master mix as follows:

A	B	C
Reagent	Concentration	Volume
Gel loading dye	6x	8 μl
Nuclease-free water	NA	16 μl

Make sure to multiply the volumes by the number of samples

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

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.

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.

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).

Submit samples for sequencing

MiSeq samples were run on a 250x250 Illumina Miseq

PacBio samples were run on a PacBio Sequel II