Here is a list of supplies and equipment that you will need to do Project GROWS, plus some suggested sources. If you have taken workshops with Fred Hutchinson's Science Education Partnership program, you can reserve the equipment you will need from them (during the SEP September equipment reservation dates).
Supplies and equipment that are used during different parts of the exercise are only listed the first time that they are used.
DNA EXTRACTION Qiagen DNA Extraction Kit Water Bath
Tissue samples Pipettors (20/200/1000)
95% Ethanol Vortex (not required)
Sharpie Pen Centrifuge (that can go over 6000g)
Dissecting Scissors and Forceps
(if students subsample fins)
1.7 ml microcentrifuge tubes
GEL ELECTROPHORESIS Agarose Horizontal Gel Box and comb
Loading Buffer Power Supply
Ladder UV Light Box
Ethidium Bromide Camera
TBE or TAE buffer
PCR PCR Beads PCR Machine
25 mM MgCl2
Primers (a forward and reverse)
Sterile distilled water
RESTRICTION DIGEST Restriction Enzyme Incubator
To obtain salmon fin tissue (chinook recommended- the PCR will not work with other species, except steelhead) contact a nearby hatchery. In Washington state, go to: http://www.wa.gov/wdfw/hat/facility.htm#Facility for a hatchery list.
To request a free DNA extraction kit, as well as Taq polymerase kit (and dNTPs) call Sonya Dobias at Qiagen Corp. at: 1-800-426-8157 Ext. 23544.
To request free DNA ladder (1 kb recommended) or restriction enzymes such as BstNI, dNTPs and other biotech supplies, call Sherry Leavitt at New England Biolabs at 1-800-632-5227 Ext 275. For free NEB catalogs (a useful reference- dial Kate at Ext. 378).
To order primers, go to http://www.fisheroligos.com. Click on “Custom Oligos.” There you will find out how to order either by phone, fax, email, or online (easiest) using a credit card or PO number. First call their customer service number 800-234-5362 to get a teacher discount and account number. When ordering online, use the default settings for amount of primer (0.05 umol), and purification (desalt). Then simply type in the sequence in the 5' to 3' direction in the box indicated. See PCR section of protocol for information on preparing the diluted primers.
To order PCR tubes, go to http://www.islandsci.com/bisland.htm where you can buy the 0.2 ml rainbow colored tubes (Cat. # IS-430R) for $39.66/bag of 1000.
To borrow equipment (and you have taken their workshop), contact the Science Education Partnership Program (SEP) at Fred Hutchinson Cancer Research Center (http//chroma.mbt.washington.edu/outreach/sep.html). Once you have taken their workshop for teachers you may borrow equipment from them.
You may borrow thermal cyclers from:
N. end: Joe Day, Lynwood H.S., 425-670-7520, DayJ@edmonds.wednet.edu
Bellevue area: Christy Brasher, Newport H.S., 425-456-7400, Shiers_Christyemail@example.com
South Sound: Dr. Peter Wimberger, University of Puget Sound, 253-879-2712, firstname.lastname@example.org.
You can also contact Washington State University's equipment loan program (http//www.sci.wsu.edu/bio/equiploa.html).
For staining DNA, use either ethidium bromide (EtBr) or SYBR Green. EtBr is highly carcinogenic and should not be handled by students- only by teachers in a prep room. You can buy a 10 mg/ml solution from Sigma (www.sigma-aldrich.com , 800-325-3010, catalogue # E1510, $34.55). Dilute to 5 ug/ml with water or running buffer and stain for 30 minutes. The stain can be poured back into a brown bottle and reused many times. Stained gels and used ethidium can be disposed of by soaking overnight in bleach, or as hazardous waste material.
SYBR green nucleic acid stain is much safer, but is much more expensive when added to staining buffer. A good alternative is to add it directly to your 10X load dye, so that after running your gel it is ready to go on the UV light box. However, you must first dilute the stock solution by 1:10,000 or else it will distort the movement of the DNA in the gel. SYBR green may also be obtained from Sigma (cat. # S9430, 0.5ml = $155.10). More information can be obtained at the manufacturer's website at http://www.molecularprobes.com.
Bio-Safe is a nice non-toxic alternative DNA stain available from BioRad, which has the advantage that the bands are visible in normal light, so no UV box or camera is required. It's also very cheap. I recommend using it at the 1:200 dilution for 1-3 hours and then destaining overnight. You can then dry the gel down on a special membrane that BioRad sells (kind of pricey), or take a picture of the gel with a regular camera. You can download the information about Bio-Safe at the BioRad website; go to Google and search for BioRad, then search their website for Biosafe. Bio-Safe is not as sensitive as ethidium or SYBR Green, but it should work fine for visualizing PCR product.
The handiest (though not cheapest) UV light box/camera setup can be found at Fotodyne. That setup is catalogue # EI-1435 adn costs about $1425 (if you tell them you’re doing GROWS they might give you a discount). Contact Brian Walsh at 1-800-362-3686, or http://www.fotodyne.com.
To borrow a thermal cycler, contact Peter Wimberger at email@example.com or Joe Day at Lynwood H.S. (DayJ@edmonds.wednet.edu), or Christy Shiers at Newport H.S. in Bellevue (Shiers_Christyfirstname.lastname@example.org).
See web page for protocols:
We have found that students seem to get more out of the project when they have more background on any and all of the topics being covered – from molecular biology and DNA replication to evolutionary processes such as migration, genetic drift, speciation and Hardy-Weinberg. We can supply readings for students that address the relevance of salmon population genetics to the “salmon problem.”
Day 1: Provide introduction to project and provide the context for why doing population genetics on salmon is interesting. (I will come to your school to do this, and bring needed supplies). [If 90 min. period, start fin clip digestions].
Day 2: Do fin clip proteinase K digests for DNA extraction.
Day 3: Finish DNA extraction. Students must work efficiently to finish in 50 min. Store DNA in freezer overnight.
Day 4: Set up and run gels of the DNA extracts (gels need to run for about 40 min. at 100V or 20 min. at 150V- in this case gel must be made with TBE in agarose and as running buffer). Stain gel (20 min. with Ethidium Bromide), look at it on UV light box, and photograph with Polaroid Camera. Note: Gels cannot be stored, so must be viewed/photographed soon after running.
While gels are running, introduce or review PCR.
Day 5: Do PCR round 1. [If 90 min. period, set up 2nd round PCR while 1st round is running (about 1 hr)].
Day 6: Do PCR round 2. Set up gel for PCR product, to be run either after class or next day (store PCR product in freezer).
Day 7: Run 2nd round PCR products on gel. While gel is running, review restriction digest concepts. Stain and photograph.
Day 8: Set up the restriction digest (1 hour to overnight for digests at 37o. 90 min. at 60o for BstN1 which is used with gonadotropin locus). I would err on the longer side to make sure that you get a complete digest. Make gels. Go over expected results of gels (possible genotypes).
Day 9: Run gels of digests, stain, photograph.
Day 10: Examine the gels. Score genotypes. Calculate whether the population is in Hardy Weinberg Equilibrium.
Day 11: Compare population to other populations (if you have done 2 populations, or use other populations on web site (still under construction). Wrap up and assessment. I will come out to your school again to do this, and pick up any loaned equipment/supplies.
NOTE: The above time-line can be condensed by a couple of days if you do the running, staining and photographing of gels outside of class.