Ciro Santilli
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More generic PCR information at: polymerase chain reaction.
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Because it is considered the less interesting step, and because it takes quite some time, this step was done by the event organizers between the two event days, so participants did not get to take many photos.
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PCR protocols are very standard it seems, all that biologists need to know to reproduce is the time and temperature of each step.
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We did 35 cycles of:
  • 94˚C for 30 seconds
  • 60˚C for 30 seconds
  • 72˚C for 45 seconds
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Figure 23. Marshal Scientific MJ Research PTC-200 Thermal Cycler. Source.
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We added PCR primers for regions that surround the 16S DNA. The primers are just bought from a vendor, and we used well known regions are called 27F and 1492R. Here is a paper that analyzes other choices: https://academic.oup.com/femsle/article/221/2/299/630719 (archive) "Evaluation of primers and PCR conditions for the analysis of 16S rRNA genes from a natural environment" by Yuichi Hongoh, Hiroe Yuzawa, Moriya Ohkuma, Toshiaki Kudo (2003)
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One cool thing about the PCR is that we can also add a known barcode at the end of each primer as shown at Code 2. "PCR diagram". This way, we were able to:
  • add a different barcode for samples collected from different locations
  • sequence them all in one go
  • then just from the sequencing output the barcode to determine where each sequence came from!
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Code 2. PCR diagram
Input: Bacterial DNA (a little bit)
... --- 27S --- 16S --- 1492R --- ...

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vvv

Output: PCR output (a lot of)
Barcode --- 27S --- 16S --- 1492R
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Finally, after purification, we used the Qiagen QIAquick PCR Purification Kit protocol to purify the generated from unwanted PCR byproducts.
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Ancestors

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