The quest for the $500 home molecular biology laboratory

By John Brunstein, PhD Molecular diagnostics and molecular biology in general are becoming more pervasive every day in a range of applications but are still seen by many as being an arcane science. Many undergraduate science curricula cover only the basics of theoretical components without exposure to laboratory practice, due to perceived cost and complexity of laboratory facilities needed. With this in mind, I recently set out on a quest to see whether a non-specialist, $500 complete molecular biology laboratory was possible. This is not a completely novel idea. Numerous previous groups have published a number of aspects of a DIY approach to molecular biology, usually aimed at teaching laboratories. These have included such things as making agarose gel boxes with graphite pencil lead as electrodes, using grocery store gelatin as a separation matrix, converting old dot-matrix printers and water baths to make thermocyclers, light-bulb driven thermocyclers, and methlyene blue as a non-toxic dye for direct visualization of DNA in gels. (For some recent examples, see references 1, 2, 3.) A recent issue of the popular magazine Wired covered the Open PCR project, which aims to bring a relatively inexpensive ($599) PCR machine into public domain use, along with such ancillary equipment as the Dremelfuge, a dremel-powered centrifuge.4 However, none of these have overcome the need for some relatively expensive and specialized equipment to conduct real experiments of interest, and in many cases have required a beyondcommon technical ability in building or modifying equipment. That this is a barrier to introducing students to a critical emerging field has been recognized by a number of groups looking to find lower-entry cost solutions suitable for educational applications.5 The challenge I set for myself had a number of criteria:

That a $500 capital equipment budget not be exceeded (Reagents and consumables were not expected to be included in this budget.) That there be little or no requirement to build or adapt devicesi.e., that standard lab equipment be used wherever possible within budget constraints That the final lab have the ability to run multiple sample types for a range of assays, with sensitivity and speed generally comparable to a real academic lab That reagents and supplies used be safe for home use That all equipment and reagents be obtained through channels available to the average person on the streetthat is, I couldnt make use of contacts in the field by calling them up and scavenging old bits of labware from under their sinks.

 

Additionally, as I went through the process of obtaining the equipment for my home molecular biology lab, I paid attention to the general availability of the various pieces of equipment; that is, was I just lucky and getting something which might on average take six months to find, or was this something one could reasonably expect to get within one to two weeks at an appropriate price range? My primary (in fact, in the end, only) resource for obtaining equipment was eBay, although I did consider and look at other potential sources. In general, and not surprisingly, I observed that local non-specialized sites such as Craigslist had little to no technical equipment but were economical choices for dual purpose consumer and lab equipment such as a microwave for preparing agarose gels. My final list of equipment obtained and prices paid (broken down into purchase price and shipping) is shown in Table 1 below. I have included current cost estimates in dollars for standard lab equipment, excluding taxes and shipping, for each item as well.

Other equipment required but not purchased, as likely readily available in everyone s house:
  

Digital camera Microwave oven Refrigerator and freezer

Table 1.
Item
PCR machine gel box pipettors transilluminator centrifuge scale pH meter Electrophoresis PS 125 ml Erlenmayer

Price
95 0 62 129.99 111 5.79 7.88

Shipping Total
45.76 0 21 6.99 40.48 0 9.16 140.76 0 83 136.98 151.48 5.79 17.04

Market price
2000 385 500 868.54 1100 500 758

39.21

8.95

48.16

1000

2.25

5.41

7.66

7.66

Totals:

453.12 137.75

590.87 7119.20

All items were purchased within a three-week period, and realistically could have been obtained within a few days had I not spent time (generally wasted, as it turned out) in trying to get items at lower costs. Most items were purchased, where possible, through a buy it now option rather than a traditional bidding auction. Importantly, all items were either new or used but sold with a warranty of full performance. In more detail, then, here s what I obtained for each listed item.

PCR Machine: A thermocycler is an absolute requirement for the home molecular lab, but fortunately there were many traditional PCR machines for sale on eBay at any given time I looked. Some knowledge of the field is useful here, as prices ranged from ridiculously cheap (a guaranteed working peltier-based instrument with an in-situ slide block went for $9.99) to outrageously expensive (a water-cooled 24-well instrument, asking $764.15; I ve used this model once in the far distant past and doubt I d have paid that for it even then). Some models are particularly common for example, the venerable Perkin Elmer GeneAmp 2400s; a search for this model alone one day showed no fewer than 33 for sale at a range of prices. After unsuccessful attempts at bidding on newer peltier-based 96-well instruments with heated lids, I opted for a Perkin Elmer 480. It was an older model, with compressor cooling and requiring the use of mineral oil to stop sample evaporation, but with a guarantee of working condition and at a price hard to pass up. Gel box: A simple plastic box with some electrodes for running agarose gels doesn t seem like it should be very expensive, even if one expects a few gel trays and a comb along with it. To my surprise, these did not appear to be readily available; average prices for a functional horizontal gel box with trays and combs were over $100. Given that this is an item that can be readily made even with next to no mechanical skills, I opted to save my budget and relax my rules on home construction slightly for this one item. Some dollar store plastic food containers, a hobby knife, a tube of silicone cement, two stainless steel bolts with nuts, and a short length of fine stainless steel wire took about 30 minutes to turn into a very serviceable and professional looking gel box, set of casting trays, and one comb. Power supply: Strictly speaking, one could dispense with getting a regular electrophoresis power supply and use seriesed 6V or 9V batteries to run the occasional gel; but given the plethora of good power supplies I saw for sale, I couldn t resist getting an old but functional BRL Model 100 power supply.

Pipettors: Another must-have component of the lab, and not one easily substituted with cheap alternatives. My total of $83, including shipping, got me a pair of good functional Biohits, a 0.5 to 10 uL and a 100 to 1000 uL model. I d have preferred also to have a third one for the missing midvolume range, but within my budget this would do, and was reasonably representative of what micropipettors were going for. Transilluminator: Most real labs use UV transilluminators as a legacy of the old days of ethidium bromide staining. Given the toxicity of this stain, I knew I couldn t use it and was initially planning to opt for SYBR Green I or SYBR Safe as my DNA stains of choice, with paired, safe, blue light illumination. Commercial blue light transilluminators are not cheap, and very few appear to be on the used market. Blue LEDs, however, are readily available and not too expensive, and I initially made my own unit. Unfortunately, the cheap strings of automotive store blue LEDs do not come with spectral specification data, and in the end the wavelength of the ones I used was a poor match to the SYBR excitation needs. Eventually I gave in and bought a used Fotodyne Foto/Phoresis I unit, to be used with Gel Red stain. This turned out to be a better choice than SYBR dyes in any case, as it s much cheaper, easily available, has excellent sensitivity, and most importantly, is classified as non-toxic and sink disposable. Centrifuge: This is arguably a requirement for the home molecular biology lab. Certainly, there are specimen types where a simple boiling lysis and use of this as assay template without any subsequent steps is feasible both in real work and in the context of a teaching laboratory. However, my goal was to have a lab capable of working on as many sample types as possible, and the existence of a variety of optimized commercial spin column-based sample preparation kits makes a microfuge a cheap way of obtaining this flexibility. My $111 plus $40.48 shipping got me a Heraeus Biofuge 13 in great condition, a variable speed microfuge with an 18 place 2 mL MFT rotor. As with other items, this came with a warranty of performance, and the detailed item description (time to achieve maximum speed, lack of

vibration, low noise) along with personal knowledge of the model made me confident I was getting a useable item. Multiple similar microfuges were available in the same price range, although some of the really newer, nicer ones went for prices approaching $600. Digital scale: Perhaps not absolutely essential, as one could buy premade buffers and prepare agarose gels by volume with kitchen measuring spoons and some trial and error. However, for $5.79 and free shipping on a buy it now option, I purchased a brand new digital microscale with 100 mg resolution and a 1 kg maximum load. At that price it came with a hardshell protective cover for the weigh pan, a soft vinyl carry case, tare capacity, the option to read in about six different units, and backlighting. Testing against a lab analytical scale showed this unit to have very good accuracy across its stated range. pH meter: Rather like the digital scale, this was non-essential but nice to have. For $17.04 I got one in a handheld form factor, digital readout, with hardshell carry case, detailed calibration instructions, and packets of reference buffer for single or dual point calibration as desired. Given what a glass pH electrode itself costs, I m not sure how these can be made so cheaply. Again, testing against a lab standard pH meter was done and showed this to perform very well. Putting it all together: Once all the equipment was there, it was time to put it to the test by attempting to sample myself and two other anonymous, consenting test subjects for a range of genomic marker types including phenotypically associated single nucleotide polymorphisms (SNPs), a deletion allele variant, and an example of variable nucleotide tandem repeats (VNTRs) similar to those used in forensics and relatedness testing. Table 2. Primers and Cycling Conditions
Primer Cycling Expected Type / Set Reference [Primer] conditions products significance

CCR5upper CCR5lower

400 nM each

95C, 2 min. 40x [ 95C, 20 sec; 56C, 30 sec; 72C, 1 min]

130 bp (wt) or 98 bp (

(32)

Deletion mutation believed to confer HIV resistance, possibly Y. pestis resistance

D1S80primer 1 D1S80primer 2

400 nM each

95C, 2 min. 40x [95C, 20 sec; 56C, 30 sec; 72C, 1 min]

Range of possible number of 16 base repeat (24 copies most common)

VNTR used for relatedness / identity testing

Amel-upper Amel-lower

400 nM each

95C, 2 min. 40x [95C, 20 sec; 56C, 30 sec; 72C, 1 min]

X derived: 106 bp Y derived: 112 bp (therefore females, single band; males, doublet band)

Gender determination on DNA samples

Rs671DAf Rs671DGf Rs671r

This report*

(Af +r) or (Gf + r) 400 nM

95C, 2 min. 40x [95C, 20 sec; 58C, 30 sec; 72C, 1 min]

195 bp product from both primer sets for heterozygotes, (Af + r) or (Gf + r) primer set respectively for A:A or G:G homozygotes

Asian Blush associated with ALDH2 enzyme G:G homozygotes show fast alcohol processing; G:A heterozygotes and A:A homozygotes show slow alcohol processing, facial flushing

*Rs671DAf: 5 GTA CGG GCT GCA GGC ATA CAa TA 3 . Rs671DGf: 5 GTA CGG GCT GCA GGC ATA CAa TG 3 . Rs671r: 5 - GCA GGT CCT GAA CTT CCA GCA G -3 . Lower case nucleotides represent intentional destabilizing mismatches to improve allele specificity.

Materials and methods Sterile water: Aquafina brand bottled water was used throughout, based on its purification method (reverse osmosis) and lack of added salts postpurification. General lab supplies: Nitrile gloves, small weigh boats, 91% isopropyl alcohol, and mineral oil were obtained at Wal-Mart. Pipette tips and 0.6 ml microfuge tubes (MFTs) were obtained via eBay from Lion International, a standard lab supplier. Two ml MFTs were obtained from a variety of sources, including individual resellers on eBay and standard lab suppliers directly. Boric acid

and sodium hydroxide were obtained from The Science Company, Denver, CO. Buccal cell collection and DNA extraction: A clean wooden sampling tool such as a flat toothpick or disposable coffee stir stick was scraped across the inside of test subject s cheek, and the cell collection portion of the sample stick broken off into a 2 ml MFT containing 200 l 0.9% domestic sea salt in water. After hand vortexing, 2 l Tide liquid laundry detergent clean breeze ActiLift was added and the sample was again gently agitated followed by incubation at room temperature for 15 minutes and floating in boiling water for five minutes. 400 l 91% isopropyl alcohol (Wal-Mart) was then added and the mixture was allowed to precipitate at room temperature for 10 minutes, followed by centrifugation, 12,000 rpm for 10 minutes. The supernatant was removed and the visible pellet allowed to air dry, followed by resuspension in 50 l water. PCR: Primer sets for a range of human genomic targets were designed by the author or taken from literature as noted in Table 2 and were obtained from Oblique Bio, Inc., (Huntsville, AL). Reactions consisted of 25 l GoTaq Hot Start polymerase (Promega Inc., Madison, WI), reaction specific forward and reverse primers at final concentrations as noted in Table 2, 1 l of buccal cell DNA extract, and water to a final volume of 50 l. Reactions were overlaid with mineral oil (Wal-Mart) and thermocycled in PE480 instrument with reaction-specific parameters noted in Table 2. Appropriate negative amplification controls were included in all reactions. Gel analysis: Agarose gels at appropriate concentrations (generally 1.5%) were prepared by melting molecular biology grade agarose (Protein Mods, Inc., Madison, WI) in 1x SB buffer prepared as per reference.6 1x Gel Red stain (Phenix Research Products, Candler, NC) was added immediately prior to gel casting. PCR products (8 l) were mixed with standard agarose gel loading dye (Qiagen Inc., Hilden, Germany) prior to gel loading. Gels were run at 100V in 1x SB buffer with a 100 bp to 3000 bp DNA size ladder (Phenix Research Products) as a standard. Gels were visualized with the described UV transilluminator and documented by handheld digital camera.

Results Clean, well resolved, readily interpretable PCR product bands were obtained for all targets examined. Negative controls (i.e., no template PCR) reliably showed no amplification, indicating that results were not arising from spurious template or amplicon contamination. Examples of SNPs, deletions, and VNTRs were thus all successfully demonstrated with the described equipment and reagents. Representative results are shown in Figure 1. The results, by test and subject, are summarized in Table 3. Discussion The author would judge the results of the quest as being successful, as a demonstrably functional molecular biology laboratory was set up within the $500 limit (not including shipping) and all other criteria were met. With regard to whether purchasing such equipment off eBay is reliable, in general, it was in this project. The author did have problems with a first power supply having an intermittent fault (this unit was resold for parts, to a buyer who was able to repair it) and did not receive delivery of the first transilluminator purchased (a refund was issued without trouble by the vendor and a second one obtained from another seller). In general, therefore, the process was relatively reliable and low-risk, as no one item was very expensive. To address whether what was done here was truly reproducible, the author has since gone back onto eBay and additionally purchased, in good working condition, a second PE480 thermocycler ($49.95), a PE GeneAmp 2400 thermocycler ($56), two 10 to 100 l pipettors ($15 and $15.50), a Pharmacia GeneQuant II UV spectrophotometer ($300), and a quartz microcuvette ($38). Increased familiarity of students with the techniques of molecular biology is desirable as these methods become more commonplace and of importance in medicine, agriculture, and other applications. A major hurdle to the inclusion of these techniques in a small college or even high school lab setting has been seen as instrument cost and assay complexity. Prior

attempts at reducing these barriers have generally required construction of makeshift equipment and/or reductions of assay performance. The widespread and increasing availability of second-hand professional laboratory equipment or inexpensive new commercial surrogates means that it is now unchallenging to set up a fully functional molecular laboratory for less than $500 in equipment costs. Coupled with the presence of sources for all reagents and supplies needed in formats that are safe for general use, the work presented here demonstrates that capacity to set up functional molecular biology teaching modules is well within the reach of even the smallest educational facilities. When coupled with outsourced PCR product Sanger sequencing available from commercial sources at prices approaching $5/reaction, the capacity of such home labs to start undertaking research of real potential scientific value such as surveys of microbial biota in unusual environments at negligible costs should not be underestimated. Similarly, the potential for setting up labs of this type for medical applications in emerging countries may be worth considering. While current best methods have moved to real-time and array-based high throughput, contamination resistant methods, the methods demonstrated here were state of the art for clinical and research molecular diagnostics in the Western world only some 15 years ago.

Figure 1. A: Results of CCR5, D1S80, and Amelogenin PCR on Subjects 1 (the author; male); 2 (male); 3 (female); and (-) negative control. L indicates lane containing 100 to 3000 bp size ladder, lower sizes as marked. B: Results obtained by PCR with rs671 A and rs671G primer sets on the authors extracted DNA; L indicates ladder; (-) is a rs671G negative (no template) control. Some final thoughts about the $500 molecular lab, in particular as being used for personal genotyping as demonstrated here. The complexities of results interpretation (not to mention lack of clinical-grade quality control on sample contamination) leave this as something to be considered only of entertainment (and possibly educational) value in the context presented here. While it is true that the general public already has access to this sort of information for a fee from services such as 23andMe or deCODEme, there remains healthy bioethical debate as to whether this has potential for harm through incomplete understanding or misinterpretation by recipients of the

data. Such risks are magnified in the hands of non-technical users, even with the best of intentions. Thus in addition to this work highlighting the potential to cheaply bring the power of molecular biology into anyone s hands for educational purposes, it should serve as a reminder to those of us professionally involved in the field that we owe society a responsibility to educate and inform as to the ethical uses, applications, and shortcomings of molecular techniques. When anyone can build a PCR lab for $500, it s past time to start educating the general population on such matters. Table 3: Observed Results Summary
Subject
1 (author; male)

CCR5
WT:WT

D1S80*
24:24

Amelogenin rs671
106:112bp G:G

2 (male)

WT:WT

24:24

106:112bp

nd

3 (female)

WT:WT

24:6

106bp

nd

(-) neg control

No product

No product

No product

No product

WT wild type allele; bp base pairs; G G allele; nd not determined. *Estimated number of repeats observed for the two alleles in the sample; 24 was the most commonly observed and corresponds to a size of 524bp (8). The author is phenotypically G:G for rs671.