Microbial ecology

1. Equipment Requirements for Microbial Ecology Practical Classes
(24/6/13)
28 students enrolled, class size cap of 35 – perhaps plan for 35 students to be
safe.
Prac 3: DNA Extraction (Aug 19, 2012):
Description:
Students will extract microbial genomic DNA from the sediment samples they collected in
Practical 1 using a commercial DNA extraction kit (MoBio Power soil DNA Isolation kit [$529
- $5.29 per extraction. Students will be given enough material for two extractions each]).
Required aliquots of each reagent will be dispensed prior to class so students each have
own stock of each reagent
Equipment required:
- Sediment sample from Prac 1
- Several boxes of rubber gloves of different sizes
- 35 x Safety glasses
- 10-20 x spatula (small – only 0.25 g of sediment collected)
- 10 x laboratory balance
- 50 x cake holders, cups/trays for weighing sediment in
- 20 x fine black textas
- 5 x small centrifuges (to fit 2 ml eppindorf tubes)
- MoBio vortex unit (JS can provide 2)
- 10 x vortexes
- 30 x 20-200ul pipettes
- 30 x 200-1000ul pipettes
- 15 x blue pipette tip boxes
- 15 x yellow pipette tip boxes
- 5 x tip disposal boxes
- 35 x plastic racks (1 per student) holding labelled aliquots from MoBIO DNA extraction kit
(for each student, in 2ml eppindorf tube):
120 ul C1
500 ul C2
400 ul C3
2 ml C4
1000 ul C5
200 ul C6

2. 190 ul of Qubit working solution, pre-mixed and in Qubit tube (Mix of ds DNA HS
assay kit reagents)
- For each student: 1 bead tube, 1 spin filter, 5 x 2ml (eppindorf tubes) collection tubes [All
from kit]
- 5 x Qubit specs
- Access to refrigerator
Post-prac requirements:
DNA samples (20 - 25 x 2ml eppindorf tubes) will need to be stored in freezer for use in
Prac. 5
Prac 5: PCR (Sept 2, 2012):
Description:
Students will perform a PCR on the DNA extracted in Prac 4 and subsequently run a gel to
visualise amplified DNA.
PCR cycle 1:34 cycle (To be programmed prior to prac – JS to provide cycling conditions)
Equipment required:
- DNA from Prac 3
- 2-4 PCR machines
- 15 electrophoresis kits/gel baths
- 3 gel illuminators
- 5 microcentrifuges that fit PCR tubes
- 35 x plastic racks (1 per student) holding 5 PCR tubes + eppindorfs containing enough to do
4 PCR reactions (sample, positive control, negative control), per reaction:
25 ul GoTaq Green Master mix
2 ul primer mix
10 ul DNA
13 ul MilliQ
 So each student will require eppendorf tubes containing:
100 ul GoTag Green Master mix
10 ul primer mix (27F, 1492R – JS to provide details)
100 ul MilliQ
15 ul positive control DNA (JS to provide DNA)
10 ul of Gel Red
Ladder (JS to provide details)
- 30 x 2-20 ul pipettes
- 30 x 20-200 ul pipettes

3. - 30 x 1 ml pipettes
- 30 x thin markers.. suitable for labelling PCR tubes
- 2-3 containers of agarose for gels
- 10-15 x 300 ml TAE for gels
-15 x 100 ml Schott bottles for mixing up gel
- 10 x 100 ml measuring cylinders for measuring TAE
- 5 x spatula big
- 4 x lab balance
- 3 x microwave
- 10 x 500 ml sterile schott bottles