Overview
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THE PROBLEM:
Some bacteria, yeasts and fungi take too long, or are too difficult, to identify in the clinical microbiology laboratory. After 3 days, the struggle needs to be overcome with effiency.
THE ANSWER:
Sequence a region of the 16S ribosome chromosome site for bacteria or the ITS chromosome site for yeast and fungi. Attostar provides reagents and procedures to get this done with a minimum of cost and effort:
The reagents:
16S Master Mix,
16S Sequencing primer,
ITS Master Mix,
ITS Sequencing primer.
Take advantage of two very nice new developments:
- First, NCBI Blast results now come with a great visual tree for results:
- Second, DNA sequencing capabilities have tremendous excess capacity.
The procedure:
- Make a suspension of the unknown bacteria, yeast, or mold in PrepMan® Ultra Sample Preparation Reagent1.
- Release the bacterial DNA by heating at 100ºC for 10 minutes.
- Release yeast and fungal DNA by vortexing with 0.5mm glass beads (provided) followed by heating at 100ºC for 10 minutes.
- Dilute the DNA.
- Use the DNA in an Attostar PCR master mix. Add AmpliTaq Gold® LD1. Do PCR.
- Treat the PCR product with ExoSAP-IT®2 at 37ºC 10 minutes and 80ºC for 10 minutes.
- Send the resulting mixture (and Attostar primer) to a DNA sequencing facility of your choice. (There are many available that will do a sequence for $8. The results are usually sent back the same day.)
- Copy and paste the results into the NCBI Blast site, and
- A few seconds later your results are available.