Difference: Protocols16SSequencing (4 vs. 5)

Revision 52011-07-15 - CraigBarnhart

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16S rRNA Sequencing to Identify Unknown Microbes

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Ever wonder what that contaminant in your culture is? Need to accurately identify an environmental isolate?

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Ever wonder what that contaminant in your culture is? Need to accurately identify an environmental isolate?

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Overview

Direct PCR of culture or isolation of DNA from culture before PCR.
PCR using universal primers.
Agarose gel to confirm PCR worked correctly.
Excise correct bands from gel and purify DNA.
Prepare and submit samples for sequencing.

PCR Reaction

For PCR we use universal primers U341F and UA1406R that amplify an approximately 1 kb stretch of rDNA and should work for nearly any bacterial or archaeal sequence [1].

primer	sequence
U341F	CCTACGGGRSGCAGCAG
UA1406R	ACGGGCGGTGWGTRCAA

The dilution of cells used in the reaction plays a critical role in the success of the amplification. Too many cells or components of certain media can inhibit PCR. For best results take a visible turbid overnight culture from a rich medium and dilute approximately 10,000-fold into the final PCR. Ideally, make a dilution series of the template cells.

The optimal dilution is somewhere between 1000 and 10,000 fold. PCR setup should include an initial denaturing step: 10min at 94 C.

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It is generally preferred to isolate the DNA from your culture before doing the 16s RNA PCR. This results in a cleaner PCR product and generally a better sequencing result.

Use the invitrogen purelink genomic kit to isolate DNA from your culture.
After DNA is isolated, use a nanodrop to find the concentration of DNA. The ideal amount of DNA for PCR is 0.1 ng/ul (final template concentration) so for a 30ul PCR reaction use 3ng of DNA.

The PCR cycle is as follows:

10 min

then 40 cycles of

94	30 sec
54	30 sec
72	1 min

then final extension of 72 for 5 min

After PCR, the products need to be run on an agarose gel to confirm correct product (around 1kb) and to excise the correct bands from the gel.

A 1.5% - 2% gel works the best for this.
After cutting the bands out of gel clean using Zymo gel extraction kit or equivalent.
To prepare DNA for sequencing follow the instructions found here: http://www.icmb.utexas.edu/core/DNA/DNA_Sequencing/FAQ-Sequencing.htm

Analysis

Use the tools at the Ribosomal Protein Database.

References

1. Baker, G.C., Smith, J.J. & Cowan, D.A. Review and re-analysis of domain-specific 16S primers. J Microbiol Methods 55, 541-55 (2003).

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