Progress was stalled for a few weeks while we tried to order the oligo primers needed for the polymerase chain reaction (PCR) from Sigma. They OK’ed the order but did not process it. We went with a different supplier, Integrated DNA Technologies and two days later the oligos were in our hands.
The oligos were dissolved and an aliquot of both oligos and the first FTA sample card were sent to MCLAB for PCR amplification and DNA sequencing on Monday 29th April. On Friday 3rd we received the results!
We tried out a number of tools for the analysis and one of the better sites which had multiple easy to use tools was DNA Subway. Take the blue line for DNA barcoding analysis! Cold Spring Harbor Laboratory also has a nice introduction to DNA Barcoding.
You can look at the results for the three samples that were successful on the following DNA subway page. Log in as guest to see the three different analysis.
We also searched the sequences with BLASTN and obtained the following results:
|1||Craterellus tubaeformis||Uncultured ectomycorrhiza (Craterellus tubaeformis) clone SWUBC461||ITS4||0.0||798||96% (596/619)||√|
|2||Laetiporus sulphureus||Laetiporus sulphureus isolate TJV95-84||ITS4||0.0||539||98% (516/525)||√|
|3||Morchella||Uncultured fungus clone WME35||ITS4||6e-25||959||72% (179/249)||✗|
|4||Morchella||Morchella punctipes isolate DD06-21||ITS1||0.0||752||99% (679/680)||√|
Unfortunately BLAST only keeps the results available for two days due to the high traffic the site experiences.
Overall our success rate was 75% on our first try! The samples were a mixture of fresh, frozen and dried samples. We are now ready to start analyzing some locally acquired samples.
There will be a presentation on the results at the next WPMC meeting along with the Oyster Mushrooms cultivation kit.