Agaricus Bisporus DNA barcoding

We’ve finally acquired enough equipment from ebay, suppliers, and junk piles to conduct our own full scale test, so to speak, of our current makeshift DIY laboratory. The whole experiment involved DNA isolation, PCR, and a gel run with visualization in order to confirm that our equipment and reagents worked as intended. We managed to complete the entire experiment using (mostly) all of our own equipment, with great results.

Our equipment/reagents we were testing:

Idaho Tech RapidCycler PCR machine
Eppendorf 5415C high speed microcentrifuge
Epoch Life Sciences plant genomic DNA isolation kit
GelGreen, agarose, TAE
Gel Electrophoresis box purchased from IO Rodeo
Electrophoresis power supply from junk pile
Fungal ID primers
Taq Mastermix

Purpose of experiment – To isolate genomic DNA from a fungus, then amplify a barcoding sequence in the ITS region of its genome via PCR, followed by verification with a gel and ultimately resulting in the PCR product being sequenced and having the results BLAST searched in the NCBI database to confirm phylogeny/species ID.

Experimental overview – A button mushroom (Agaricus Bisporous) was purchased from the store for 16 cents, and a plant genomic DNA prep kit from Epoch was used on a small sample from both the cap and the stem of the mushroom (freshly cut in half).

We used two different pairs of primers: ITS1 / ITS4 and NLB4 / NSI1

These primers target an area of DNA (known as rDNA) which codes for ribosomal RNA comprising the large and small ribosomal sub-units (LSU, SSU), and the internal transcribed spacer (ITS) which has become a “standard” for barcoding and species identification of fungi.

NSI1 (forward) GAT TGA ATG GCT TAG TGA GG
NLB4 (reverse) GGA TTC TCA CCC TCT ATG AC
ITS1 (forward) TCC GTA GGT GAA CCT GCG G
ITS4 (reverse) TCC TCC GCT TAT TGA TAT GC

We expected products of around ~700 for ITS and ~900-1kb for NLB/NSI but these can vary for different mushroom varieties (ie. basiodiomycetes vs ascomycetes etc)

We tested our PCR machine, an Idaho Technologies Rapid Cycler vs a peltier effect based machine. The Idaho Tech machine uses a halogen lamp and a tornado-like air circulation effect for fast ramp times. Neither of the machines had heated lid capabilities, so mineral oil was used in each PCR reaction to avoid evaporation.

After gDNA isolation, PCR reactions were setup as follows for a final reaction volume of 25 uL in 200uL thin walled plastic PCR tubes.

10uM forward primer 0.5uL
10uM reverse primer 0.5uL
Template DNA 2uL
OneTaq 2x Master mix 12.5 uL
ddH2O 9.5 uL

reagents

Our PCR program was as follows:
94C for 30s
54C for 45s
72C for 45s

30 cycles (we wanted 35 but time didn’t allow for it)

Even at 30 cycles and those program times, the Idaho Tech machine finished ~30-45 minutes before the peltier machine, boosting confidence in the PCR machine purchased off ebay for $50.

10uL of our PCR product was run on a 1% agarose gel pre-stained with GelGreen. Product sizes were compared to 1kb NEB ladder, and single bands were observed for all wells, minus the presence of some primer dimers.

Optimization of PCR is needed, but this is a great first step which proves that with a little ingenuity and some patience in acquiring decent equipment on ebay, one can do some decent molecular biology on short funds.

The samples are being prepped for sequencing and another post will follow giving our findings.

gel

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