<noautolink> ---+ 16S rRNA Sequencing to Identify Unknown Microbes Ever wonder what that contaminant in your culture is? Need to accurately identify an environmental isolate? <br> Overview <br> * Direct PCR of culture or isolation of DNA from culture before PCR. <br> * PCR using universal primers. <br> * Agarose gel to confirm PCR worked correctly. <br> * Excise correct bands from gel and purify DNA. <br> * Prepare and submit samples for sequencing. <br> ---++ 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. <br> 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. <br> Use the invitrogen purelink genomic kit to isolate DNA from your culture. <br> 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. <br> The PCR cycle is as follows: <br> | 94 | 10 min | then 40 cycles of | 94 | 30 sec | | 54 | 30 sec | | 72 | 1 min | then final extension of 72 for 5 min <br> 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. <br> A 1.5% - 2% gel works the best for this. <br> After cutting the bands out of gel clean using Zymo gel extraction kit or equivalent. <br> To prepare DNA for sequencing follow the instructions found here: http://www.icmb.utexas.edu/core/DNA/DNA_Sequencing/FAQ-Sequencing.htm <br> ---++ Analysis Use the tools at the [[http://rdp.cme.msu.edu/][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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Topic revision: r5 - 2011-07-15 - CraigBarnhart