Mike Deyholos, 12 April, 2000.
1.
Who should I contact about microarraying in the Galbraith lab?
2.
What is the cost of producing and analyzing a microarray?
3.
How many spots can fit on a slide?
4.
How long does printing take?
5.
Who will do the printing?
6.
What do users need to provide for printing ?
1. Q: Who should I contact about microarraying in
the Galbraith lab?
A: After reading this document, users should
contact Dr. Galbraith to discuss their project. Technical questions should be directed to Dom Decianne, Mike
Deyholos, or Betsy Pierson (626-5460
or 621-9213; e-mail addresses from the
Galbraith Lab webpage). Scheduling of
printing should be done through Dom Decianne.
2. Q:
What is the cost of producing and analyzing a microarray?
A: The total cost is the sum of sample
preparation, printing, and hybridization expenses. Our lab spends approximately $0.85 per sample to amplify and
purify DNA suitable for printing. Taq
polymerase is the most expensive component of the sample preparation cost; and
your cost may vary. Printing costs are
detailed below (see below: What do users need to provide for printing ?), but
range between $0.50 and $12 per slide, plus $139 per pin. Finally, each 2-channel hybridization will
cost you $20 - $30, due mostly to the price of fluorescent dyes. Note that these estimates include only the
cost of consumables, and do not allow for failures, replications, etc.
3. Q:
How many spots can fit on a slide?
A: We recommend printing with a 225mm
(center-to-center) spacing. At this
pitch, 6400 spots will easily fit under the area of a standard (22x 22mm)
coverslip. Close to 20 000 spots can
therefore be placed on the entire area of the slide. When necessary, spots can be spaced as close together as 200mm.
4. Q:
How long does printing take?
A: This depends on the number of slides, the
number of samples, the number of replicate spots per sample, and the number of
pins used. We can print with 1, 4, or
16 pins. Printing goes faster with
more pins, but because a 4-pin print will necessarily cover an area of at least
12x12 mm, users with a small number of samples ( >300 ) generally prefer to
print with a single pin. As a
guideline, using 4 pins, it takes 35 min. to print 100 slides from a single
96-well plate, with 3 replicate spots per sample.
5. Q:
Who will do the printing?
A: Galbraith lab personnel will calibrate and
program the printer for you. After
that, you will need to stay by the instrument for the duration of the print run
in order to insert new sample plates, and to monitor chamber humidity. Print runs can be paused overnight, and for
meals, etc.
6. Q:
What do supplies do users need to provide?
A: You will need to bring your DNA samples,
printing pins and slides:
Purified DNA
should be suspended in 7-10 uL 2X SSC.
The concentration of DNA in this volume should be at least
400ng/uL. Samples must be presented in
96-well, V-bottom plates ( Starstedt # 82.1583). Other brands and types of plates are not acceptable, as the
instrument is calibrated to use the specified plates.
To ensure the
best print quality, we ask all users to purchase their own printing pins. We can make exceptions to this rule for very
small runs. Pins cost $139 each, and
should be purchased from Majer Precision Engineering (http://www.majerprecision.com/pins.htm ; brand name Microquill
2000). Pins from other manufacturers
cannot be used with our instrument at the current time.
To increase
hydrophobicity and DNA-to-slide binding, microscope slides for microarraying
are coated with compounds such as
poly-L-lysine, aminoalkylsilanes, or aldehyde-silanes. We can print on any type of coated slide
that you bring.
Slides coated
with poly-L-lysine are inexpensive (~$0.50 ea.), and can be made in the
laboratory (http://cmgm.stanford.edu/pbrown/protocols/1_slides.html ), or
purchased from suppliers such as Sigma (brand name: Poly-Prep). Poly-L-lysine slides work well for most
purposes, but some users prefer to buy aminoalkylsilane or aldehyde-silane
slides made specifically for microarraying.
These range in price from $7 to $12 each and can be purchased from a
variety of manufacturers, including Sigma, Corning, and Telechem
(http://www.arrayit.com). Note that Sigma
sells two types of silanated slides:
Silane-Prep and Array-Prep; only the Array-Prep slides are designed for
microarraying.
Whichever type
of slide that you use, you should scan a small sample of slides from each
batch, PRIOR TO PRINTING, to ensure that there is no detectable background
fluorescence.
Note that the
first 5-10 slides in any print run often have printing defects (e.g. irregular
spots), so you may want to use your cheapest slides in these positions.