BioHackerLab/Experimental
Experiments performed at Bitraf.
Innhold
05 Jul 2016 - Bitraf PCR #1
10 Jul 2016 - Bitraf PCR #2
11 Jul 2016 - Bitraf PCR #3
PCR was performed using the same PCR setup and program as July 10 except for the selection of templates and template amounts. The following templates and template amounts were used:
- #1/HS1A: 1 uL yeast sample #1. (S. Cerevisiae [1])
- #2/HS1B: 2 uL yeast sample #1. (S. Cerevisiae [1])
- #3/HS2A: 1 uL yeast sample #2. (Brettanomyces? [1])
- #4/HS2B: 2 uL yeast sample #2. (Brettanomyces?[1])
- #5/P1: 1 uL S. cerevisiae (Idun tørrgjær. Template prepared 5 july stored in freezer)
- #6/P2: 2 uL S. cerevisiae (Idun tørrgjær. Template prepared 5 july stored in freezer)
- #7/NT: Reaction mix and primers only, no template.
The PCR samples were moved to freezer after completion of the PCR run.
[1]: Liquid cultures provided by Heikki. To prepare template, 50 uL liquid culture was incubated at 98C for 10 min in OpenPCR.
Expected results/bioinformatics analysis
Reference sequence:
One of the yeast samples was described as Brettanomyces. The most relevant organism in this genus appears to be Brettanomyces bruxellensis. The organism overview page at http://www.ncbi.nlm.nih.gov/genome/11901 lists Brettanomyces bruxellensis CBS 2499 (assembly Dekkera bruxellensis CBS 2499 v2.0, Assembly accession no. GCA_000340765.1) as the representative genome for this organism.
BLAST search:
A BLAST search was performed in the GCA_000340765.1 assembly with the expected PCR product sequence ( for amplification with primers ITS1 and ITS4 from S. cerevisiae, as determined by in silico PCR (link). The search returned 15 hits ("Sequences producing significant alignments". The longest 100% matching sequence stretch was 17 bp long, having an Expectation value ("E value"; roughly, the probability of observing the result by chance) of 5.1. The hit with lowest Expectation value and the only hit with an E value below 1, is a 34bp stretch with 28/34(82%) matching nucleotides and an E value of 0.42.