Manual Glass Slide Replicator Wash and Blot Stations

MANUAL GLASS SLIDE MICROARRAYER

We are proud to introduce the first economical and practical way to make microarrays on glass slides.
Now you can make your own DNA or protein microarrays without buying an expensive automatic spotter
machine. Whether you want to make large arrays on glass slides, work out new protocols for your robotic
Glass Slide Arrayer or just Q/C new DNA preparations, this simple and flexible system allows you to
tackle both large and small jobs without major setup hassles. Now you can have:

· Convenience
Print your own microarrays anytime from 96 or 384 well plates

· Versatility
Can spot your samples as duplicates, triplicates or more

· Enhanced Features
Print up to 768 spots per array. Print duplicate slides at the same time.

· Spot Size
~500 microns in diameter on silanated slides and 780 microns on Epoxy coated slides

· Spot Pitch
1,125 microns on horizontal axis and 750 microns on the vertical axis, can change pitch easily for different
spot sizes by skipping rows or columns

· Accessories Available
Indexing System for 96 and 384 well source plates to simplify arraying and enhance spot size reproducibility.
Replicator pin wash station to enhance arraying speed.

Our Floating Pin Replicator can be fixtured with either 2 rows of 4 pins (8 total) on 9 mm centers for
96 well microplates (87v-478) or 4 rows of 8 pins (32 total) on 4.5 mm centers for 384 well microplates
(87v- 478A). When used in conjunction with the unique Glass Slide Indexing System (87v-470) (patent
pending) this dual slide system places 768 spots ~500 microns in diameter, onto a grid 18 mm wide and
36 mm tall on two separate slides. The ability to index two slides at the same time facilitates your ability
to make duplicate slides. This also saves the time and effort required to wash the pins between slides. The
pitch or center to center spacing between the spots is 750 microns on the Y axis and 1,125 microns in the
X axis. This array will accommodate eight, 96 well microplates or two, 384 well microplates. However,
you can use as few spots as your library requires or as spot size dictates. We have found that our unique
replicator pins carry ~ 6 hl and leave a ~3 hl drop on the slide which produces a 500 micron diameter spot
on silanated slides. This spot size allows a 24 by 32 array or 768 spots/array, while on epoxy coated
slides, the same 3 hl drop produces a spot of ~780 micron in diameter, so the highest density is 384 spots/
epoxy coated slide.

Q: How can I do quantitative studies on high density arrays made on membranes?
A: Although there are several image analysis programs that may be used to determine pixel intensity and total pixel quantitative,
we have found that the ImaGene™ image processing software from BioDiscovery, Inc., provides a very simple and compatible
system. See image of segmented ImaGene display.

The Glass slide above illustrates the 768 spotting pattern on a
silanated glass slide

The glass slide (right) is an epoxy coated slide spotted
with 5 mm 24mer oligonucleotide with a 3' amine linker
and a 5' Cy5 label in 50% DMSO. The Slide was scanned
by Axon Instruments, Inc. on their GenePix 4000 Scanner. The GenePix 3 software was used to determine a mean spot diameter
of 780 microns with a CV of 5.5%.

ACCURACY AND PRECISION DATA FROM THE SLIDE ABOVE
SPOT DIAMETER

Position	Mean	SD	%CD	Max	Min
Pin 1	793.1	42.7	5.4	920	700
Pin 2	786.7	42.1	5.4	910	700
Pin 3	789.2	40.5	5.1	890	710
Pin 4	786.3	35.4	4.5	870	730
Pin 5	787.5	39.1	5.0	910	710
Pin 6	757.9	41.4	5.5	870	690
Pin	7 795.8	41.7	5.2	930	730
Pin 8	745.2	37.4	5.0	840	690
Total	780.2	43.3	5.5	930	690

We have also obtained
data using SigmaScreen
Silanated slides with a
24mer 5uM Oligo with a
5'-Cy5 label spotted in
50% Array It and 50% 15 mM Sodium Phosphate
buffer and scanned on an Applied Precision
(API) Array WoRx Scanner. BioDiscovery
software was used to determine a mean signal
area of 7,914 with a CV of 5.7% and a mean signa of 40,434 with a CV of 4.3%.

ALDEHYDE (SILANATED) SLIDE WITH AMINO/CY5-LABELED
OLIGO, 768 spot array, 5' Cy5 and 3'Amino linker

Minimum	3142
Maximum	41410
Min	30302
Std Dev	3707
%CV	12.2

Factors Affecting Pin Delivery Volumes

Pin diameter
Surface tension of the liquid being transferred
Surface tension of the pin
Speed of removal of pin from source liquid
Speed of pn striking recipient dry plate
Depth to which the pin is submerged in source plate
Depth to which the pin is submerged in recipient plate
Volume of slot in pin
Surface tension of the dry plate

Q: What is the accuracy of delivery using slot pins?
A: In tests transferring 5 ml of nitrophenol from one microplate to another using either a 12 channel pipette or a 87V-408S5 5 ml
Slot Pin Replicator, we determined that the accuracy of delivery was much better with the Slot Pin Replicator. The Coefficient of
Variation for the Slot Pin Replicator varied between 2% and 5%. The 12 channel pipette consistently had a much higher Coefficient
of Variation than the Slot Pin Replicator, varying from 8% to 22%.

The mean color intensity and Coefficient of Variation for each row of 12 wells are as follows:

Row A - pipette 0.312 +/ - 16%, ......slot pin 0.301 +/- 5%
Row B - pipette 0.316 +/- 9%,..........slot pin 0.285 +/- 3%
Row C - pipette 0.337 +/- 16%,........slot pin 0.286 +/- 2%
Row D - pipette 0.315 +/ - 8%,..........slot pin 0.292 +/- 3%
Row E - pipette 0.303 +/- 22%,........slot pin 0.295 +/- 2%
Row F - pipette 0.358 +/- 16%,.......slot pin 0.294 +/- 3%
Row G - pipette 0.399 +/- 11%,.......slot pin 0.291 +/- 2%
Row H - pipette (no data)..................slot pin 0.296 +/- 2%

Summary of Slot Pin to Slot Pin Delivery Precision

Pin Diameter (mm)	Volume of Slot	Mother Well Solvent	Recipient Well Solvent	Label	CV
1.58	5 uL	Aqueous	Aqueous	Methyl Orange	3.7%
1.58	2 uL	Aqueous	Aqueous	Methyl Orange	4.9%
0.457	50 nL	Aqueous	DMSO	FITC	3.2%
0.457	50 nL	ETOH	DMSO	FITC	3.4%
0.457	50 nL	Aqueous	DMSO	FITC	4.0%
0.457	10 nL	ETOH	DMSO	FITC	2.7%
0.787	100 nL	Aqueous	Aqueous	Horseradish Peroxidase	1.6%
0.787	200 nL	Aqueous	Aqueous	Horseradish Peroxidase	1.9%

Q: How can I transfer larger volumes using pins?
A: By placing grooves or slots in the pins that pickup liquids via capillary action. The volume of liquid picked up is directly determined by the size of the groove or slot in the pin.

Our Grooved Pins transfer from 3 ml to 20 ml. They are used to transfer liquid to liquid between a mother plate and a daughter
plate. Both plates must have a liquid level high enough to cover the grooves.

Our Slot Pins transfer from 5 hl to 25 ml. They are used to transfer liquid to liquid between a mother plate and a daughter plate
and to make blots on membranes and agar surfaces. Slot pins also have the advantage of being able to transfer to and from mother and daughter plates that have very low volumes as the slot is in the bottom of the pins and will wick up very tiny volumes in the bottom of the wells if the slot pins are cleaned with the 87V- 110 Surfactant or have been dipped in ETOH and flamed.

87V-470 Glass Slide Indexer has the 8
place horizontal X axis indexing holes
and the 12 place vertical Y axis indexing
holes with their respective pins.
Changing an index position is as simple
as moving the pin to the next hole.
This ingenious indexing system is capable
of exacting precision and accuracy.
Our unique indexing system also
guards against the accidental misplacement
of the Indexing pin into the
wrong indexing hole. The two sets of
guide pins are used to locate the 87V-
478 and 87V- 478A Arrayers onto the
two glass slide bays of the 87V- 470
Glass Slide Indexer. The guide pins
locate the Arrayers by fitting into holes
in the bottom of the Arrayers. This
provides a very rapid yet simple and
easy way to locate the Arrayers. Another
"fool proof" system incorporated
into the design is that the pair of guide
pins have different diameters thus preventing
the Arrayer from being placed
in the slide bay in the reversed position.
87V-478

In addition to spotting on glass
slides or chips our Glass Slide
Arraying system will also spot
onto the new FAST microporous
polymeric surface system produced
by S&S on glass slides.
The photo on the right illustrates
spotting on this substrate. For
spotting on to the FAST microporous
polymeric surface we re commend
using our smooth tipped
pins the FP1. They are similar to
the FP1S6 in diameter and length
but carry a much larger 20 hl
hanging drop to the membrane.

MANUAL GLASS SLIDE MICROARRAYER SYSTEM

87V-478	8-Pin Replicator	For 96 well microplate (9mm centers) Size: 8.4W x 2D x 11H cm Weight: 167 gr Constructed of delrin with stainless steel pins Comes with 8 fixed pins (87V-478FP1S6N) plus 4 extra replacement pins.
87V-478A	32-Pin Replicator	For 384 well microplate (4.5mm center) Size: 8.4W x 2.5D x 11H cm Weight: 230 gr Constructed of delrin with stainless steel pins Comes with 32 fixed pins (87V-478FP1S6N) plus 4 extra replacement pins.
87V-470	Manual Glass Slide Indexing System	For 25 x 75mm with 1 mm thick or 1 x 3” slides The spring-loaded locator will allow irregular slides up to 25.9mm (1.02”) wide and 76.36mm (3.007”) long to be loaded. Thinner or thicker slides can also be accommodated. Size: 15W x 13D x 4H cm Weight: 1.078 kg
87V-475	Manual Glass Slide Replicator Wash and Blot Station	This item is used to facilitate and speed the washing of the replicator pins between Specimens. Any fluid can be used in the p reservoirs. However, for most nucleic acid applications the first wash reservoir usually contains 5% bleach, the second reservoir contains distilled water, the third reservoir contains distilled water and the fourth reservoir contains 100 ETOH. The ETOH is evaporated off using a hot air blower (hair dryer). Each reservoir has its own blotting station and the ins are blotted prior to submerging in the next reservoir. Also the depth of fluid in each reservoir is slightly greater sas you progress from left to right so that wash solution goes farther up the pin shaft each time to insure a through washing operation. Guide pins on both the wash and blot stations ensure that the pins are properly aligned and protected from damage.
87V-478FP1S6N	Replacement Replicator Pins	457 micron diameter, exposed tip 17mm long with 6 nl slot, deliver –3 nl to glass slide.
87V-478FP1S6NT	Replacement Replicator Pins	457 micron diameter, exposed tip 28mm long with 6 nl slot, deliver –3 nl to glass slide.
87V-478FP1	Replacement Replicator Pins	457 micron diameter, exposed tip 17mm long with smooth tip, deliver – 20 nl to membrane
87V-110	Surfactant	If you are transferring DNA, cDNA or other materials in solution with high surface tension Surfactant treatment of the pins will greatly increase the precision of delivery (30ml/bottle)
87V-522	Lint Free Blotting Paper	Lint free blotting paper is important in the cleaning all of our slot pin replicator pins and glass slide replicator pins as lint particles in the slots will interfere with the loading and unloading of liquid in the slots. Cut for replicators (Size: 8 x 11.5 cm)
87V-522A	Lint Free Blotting Paper	Lint free blotting paper is important in the cleaning all of our slot pin replicator pins and glass slide replicator pins as lint particles in the slots will interfere with the loading and unloading of liquid in the slots. Cut for Glass Slide Replicator Wash and Blot Station (Size: 5 x 31 cm)

One of the problems that has arisen in spotting DNA onto membranes or glass slides has been that the spotting solutions often
have high surface tension which results in variable volume hanging drops and therefore, variable spot diameters. We had first
noticed that distilled water has a very high surface tension and when just used with dye (food coloring) to make practice arrays
this mixture produced very irregular spot sizes. We also found that the irregular spot size can be eliminated by adding a detergent
such as Tween 20 or proteins, carbohydrates or carrier DNA to the solution. However there are many protocols where it
is not possible to add these surface tension lowering compounds to the solution.
Another way to address the problem of high surface tension liquids is to clean the pins with a special solution which cleans the
pin and changes the surface tension of the pin thus compensating for the high surface tension of the liquid. Just dipping the pins
into this solution briefly cleans the pins and changes the surface tension of the pin. The pins are washed in water and ethanol to
remove any residual solution and used right away. This pin cleaning treatment results in very uniform dispense volumes.

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