abi PRISM 377 User manual

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ABI P
RISM
®
377 DNA Sequencer
96-Lane Upgrade
User’s Manual
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© Copyright 2000, Applied Biosystems
For Research Use Only. Not for use in diagnostic procedures.
ABI PRISM and the ABI PRISM design, AmpliTaq, GeneAmp, and GeneScan are registered trademarks of PE Corporation or its
subsidiaries in the U.S. and certain other countries.
ABI,AFLP Plant Mapping,AmpF l STR,AmpF l STR Profiler,AmpF l STR Profiler Plus,ApliTaq Gold,Applied Biosystems, BigDye
Primer, BigDye Terminator, and True Allele, are trademarks of PE Corporation or its subsidiaries in the U.S. and certain other
countries.
All other trademarks are the sole property of their respective owners.
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Contents
i
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1
Upgrade Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2
Important Upgrade Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3
Kit Configurations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4
2 Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1
Preparing Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2
Setting Run Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-5
Loading Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-7
3 Software and Firmware . . . . . . . . . . . . . . . . . . . . . . 3-1
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-1
Software. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-2
Firmware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-5
4 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-1
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-1
Troubleshooting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-2
A Filter Set/Dye Combinations . . . . . . . . . . . . . . . . . .A-1
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ii
B Two-Pitch, Eight-Channel Loader Suppliers. . . . . B-1
Supplier Information Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-1
C Part Numbers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-1
ABI P
RISM
377 DNA Sequencer Parts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-1
ABI P
RISM
DNA Fragment Analysis Kits and Reagents . . . . . . . . . . . . . . . C-3
ABI P
RISM
DNA Sequencing Kits and Reagents . . . . . . . . . . . . . . . . . . . . . C-8
User’s Manuals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-12
Part Number Updates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-12
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Introduction 1-1
Introduction 1
Overview
About This
Manual
This manual describes the enhancements to the ABI P
RISM
®
377 DNA
Sequencer included in the 96-lane upgrade.
Be sure to place this manual in your ABI P
RISM
377 DNA Sequencer
User’s Manual .
Technical Support
Contacts
For technical support contact information refer to the ABI P RISM 377
DNA Sequencer User’s Manual .
In This Chapter
The following topics are covered in this chapter.
Topic See Page
Upgrade Overview 1-2
Important Upgrade Notes 1-3
Kit Configurations 1-4
1
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1-2 Introduction
Upgrade Overview
Product Overview
The ABI P
RISM
®
377 DNA Sequencer 96-lane Upgrade Kit enhances
the capabilities of the 377 DNA sequencer to support up to 96 lanes for
both Sequencing and GeneScan applications.
Key Features
The following list provides an overview of the key features of the 96-lane
upgrade.
♦ Increased scan window by 20%, allowing additional lanes to be
added without losing sensitivity or increasing scan time
♦ Increased number of data collection to 480 channels, allowing data
collection of three channels plus two-channel separation per lane
♦ Improved Neural Net Tracker, decreasing labor to process gels
♦ Increased comb thickness in loading area to 0.4 mm while using
0.2-mm gel for electrophoresis, causing no change in run time
♦ Improved comb durability and geometry, allowing easier loading of
volumes up to 1.5 µL
♦ Added position-based CCD integration with time scaling, allowing
collection while accelerating the stage, which minimizes noise
♦ Upgraded instruments still run 36-, 48-, and 64-lane gels
Hardware
Required
The following hardware is required to upgrade to 96-lane capability.
♦ ABI P
RISM
377-36 or 377XL DNA Sequencer
♦ Macintosh
®
computer with the following specifications
– Power PC processor
– 32 MB RAM (RAM modules supplied if required)
– Mac OS 8 (supplied in kit)
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Introduction 1-3
Important Upgrade Notes
Matrices
To ensure data quality, we strongly recommend rerunning matrices at
installation and semiannually for applications where matrices may be
critical for optimal signal-to-noise ratio (e.g., heterozygote detection and
any GeneScan application).
Combs
The 96-lane run mode only supports the use of a shark’s-tooth comb.
Clamps
The 96-lane plates and casting combs require 10–12 lbs.clamping
pressure.To prevent well leakage, only use clamps that meet this
requirement. Use our stainless steel “bulldog” clips (P/N 4305386) or
measure other clamps with a force gauge.
Mac OS 8
Extension
Conflicts
There are known conflicts with some of the Mac OS 8 extensions. It is
important to turn off these extensions before beginning any 96-lane run.
From the Extensions Manager window turn off the following extensions:
♦ Open Tpt Modem
♦ Open Tpt Remote Access
♦ Open Tpt Serial Arbitrator
Computer Notes
Can Run on Any Macintosh Computer
96 lanes can be run on any Macintosh computer supplied with the 377
instrument.
Processor Speed
The processor speed does not impact Collection, but it does impact the
speed of analysis.
Hard Drive Disk Space
The hard drive must have enough disk space to hold a 70 MB gel file.
A CD Drive Is Required to LoadAnalysis Software
Analysis will work on any Macintosh computer. However, the 7100
Macintosh computers supplied with the 377 instrument were not ship-
ped with a CD drive, which is required to load the Analysis software.
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1-4 Introduction
Kit Configurations
Kit Configurations
Table
The following table lists the components and quantity of components
included in ABI P
RISM
®
377 DNA Sequencer 96-lane upgrade kits.
Component
(Quantity) Part No. (P/N)
Kit Contents
377-96-
66/80B 377-96- 90B 377-96- 90C 377-96- 120C 377-96- C377-96- XL
Kit, stepped
plates, 36-cm,
pair of spacers
a
4305693 222222
Disk, 377
Collection s/w
v. 2.5
4305535 111111
Manual, user’s
96-lane upgrade 4305423 111111
Clamps, glass,
2-in. 4305386 333333
Comb, 100-well,
shark’s-tooth
cast, 0.4-mm,
1.8-mm center
4305385 222222
Kit, EEPROM,
96-lanes —111111
PCA, tested
16 MHz, 377XL —1 1 ————
Upgrade 8 MB
Power Mac RAM
SIMM
—2 —————
Upgrade 8 MB
Power Mac RAM
DIMM
——221——
Mac OS 8 — 111111
Seq Anal 3.2
b
—111111
GeneScan 3.0
b
—111111
a. Spacers are 48 cm and need to be cut to size before use.
b. As licensed.
Note The 96-lane upgrade also includes hardware modifications that will be made by the service engineer at
installation.
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Gels 2-1
Gels 2
Overview
In This Chapter The following topics are covered in this chapter.
Topic See Page
Preparing Gels 2-2
Setting Run Conditions 2-5
Loading Gels 2-7
2
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2-2 Gels
Preparing Gels
Pouring Gels
To pour gels for use on the ABI P
RISM
377 96-lane DNA sequencer:
Step Action
1 Cast the gels as instructed in the ABI P
RISM
377 DNA Sequencer
User’s Manual. Use 0.2-mm spacers (P/N 401837) and a 0.4-mm
96-lane casting comb (P/N 4305385) with the new stepped front
plate (P/N 4305384).
! WARNING !
CHEMICAL HAZARD. Acrylamide and Bis-
Acrylamide are both poisons, neurotoxins, irritant,
carcinogens, and possible teratogens. Acrylamide and Bis-
Acrylamide sublimes (the solid releases toxic vapor) and is
harmful if swallowed, inhaled, or absorbed through the skin.
Effects are cumulative.When handling, always wear personal
protection (i.e., lab coat, safety glasses, and chemical
resistant gloves) and use in a well ventilated area.Thoroughly
clean surfaces subject to contamination (i.e., binder clips,
combs, and glass plates).
2 Clamp the gels as shown below. Use three stainless steel “bulldog”
binder clips (P/N 4305386) on the top.
IMPORTANT To prevent well leakage, the 96-lane plates and
casting combs require 10–12 lbs.clamping pressure.
IMPORTANT Be careful not to damage the teeth of the comb
when attaching the clamps.
End clamps align with
front plate notch Clamping pressure
on casting comb
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Gels 2-3
Preparing a Gel
To prepare a gel for a run:
Step Action
1 Let the gel polymerize for at least two hours.
2 Remove the clamps from the gel. Leave the casting comb in place
until you are ready to insert the comb.
3 Rinse the plate thoroughly with dH
2
O and let dry.
IMPORTANT Carefully clean the read region of the gel.
4Slide out the casting comb without bending it.
IMPORTANT Do not pry the casting comb.
5Using a razor blade, scrape off all excess acrylamide from the glass
in the loading area.
6Using a squirt bottle, rinse the loading area with dH2O and dry with
at lint-free tissue.
7Using a syringe, add 1x TBE in to the loading area.
Note Adding TBE eases the insertion of the comb.
IMPORTANT Be very careful not to introduce bubbles.They
are very difficult to remove once the comb has been inserted.
8If necessary, clean the shark’s tooth comb with dH2O and a lint-free
tissue.
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2-4 Gels
9Carefully insert the comb into the gel.
a. Carefully align the center registration line on the comb with the
registration mark on the back plate.
b. Slide the comb between the plates.
IMPORTANT To avoid bending or breaking the teeth of the
comb, ensure all teeth enter the space between the plates at the
same time.Do not force the comb into the gel because the teeth will
bend, causing leaking.
c. Continue to slide the comb down until the tips of the teeth just
touch or slightly depress the surface of the gel.
d. Teeth should just barely indent the surface of the gel. If the
surface of the gel is not completely flat in the loading region,
insert some of the teeth below the surface of the gel (up to
0.5 mm) so that all of the teeth touch the gel surface.
e. If a tooth has penetrated the gel surface do not attempt to
withdraw the comb.This will cause sample to leak into adjacent
wells.
10 Place the gel and the cassette in the 377 instrument.
Note For instructions on setting up the 377 instrument for a run
refer to the ABI PRISM 377 DNA Sequencer User’s Manual.
Step Action
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Gels 2-5
Setting Run Conditions
Selecting a Run
Mode Use the following table to select the type of comb to use based on the
number of lanes you are running.
Note The correct run mode is automatically chosen when the number of
lanes is selected.
Note It is possible to run a gel of any number lanes in 96 Scan mode.There
will be the same number of data collection points per lane, but there will be an
area of blank space to the left and right of the samples due to extra scan width.
However, the Neural Net Tracker has been trained using gels run according to
the default parameters.(For example: 48-lane gels run in XL mode and 36 lane
gels run in Full Scan mode.) Any deviation from the default is likely to confuse
the tracker resulting in mistracked lanes.
Setting Run
Conditions To set gel run conditions:
No. of Lanes Comb Run Mode
24 Shark’s-tooth Full Scan
24 Square-tooth Full Scan
32 Shark’s-tooth Full Scan
34 Square-tooth Full Scan
36 Shark’s-tooth Full Scan
36 Square-tooth Full Scan
48 Shark’s-tooth XL Scan
50 Square-tooth XL Scan
64 Shark’s-tooth XL Scan
66 Square-tooth XL Scan
96 Shark’s-tooth 96 Scan
Step Action
1Open the 377-96 Collection software.
2Prepare a sample sheet as described in the ABI PRISM 377 DNA
Sequencer User’s Manual.
IMPORTANT Preparing a sample sheet prior to the run is
required for optimal tracker operation.
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2-6 Gels
3Select a new GeneScan or Sequencing run.The Run window is
displayed.
4Within the Run window perform the following:
a. Select 96 from the Lanes pulldown menu.
Note The correct run mode is then automatically selected.
b. Select the plate check Pre-run and Run modules that
corresponds to your desired filter set from the appropriate
pulldown menus.
c. Select the proper instrument file (matrix) for your run.
IMPORTANT The tracker will not function unless the matrix file
was selected before starting the run.
d. Select the proper sample sheet.
5Perform the plate check, prerun, and run procedures as instructed
in the ABI PRISM 377 DNA Sequencer User’s Manual.
Step Action
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Gels 2-7
Loading Gels
Loader Options The following loaders can be used to load a 96-lane gel.
♦Fixed-pitch, 10.8-mm loader and a plate rack holding micro-amp
tubes spaced 10.8 mm apart
♦P-10 microliter pipet with a flat loading tip
♦Single-barrel syringe with 0.2-mm or 0.3-mm needles
♦Two-pitch, eight-channel loader
Note For reasons of loading speed and accuracy, Applied Biosystems highly
recommends using a two-pitched, eight-channel loader to load 96-lane gels.
Two-Pitch, Eight-
Channel Loader The following schematics depict generic two-pitched, eight-channel
loaders in their closed position.
Note For specific vendor information, see “Two-Pitch, Eight-Channel Loader
Suppliers” on page B-1.
Distance between needles:
♦9 mm in closed position
♦10.8 mm in open position
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2-8 Gels
Suggested
Sequencing Load
Volumes
The following table lists the suggested load volumes and sample
resuspension volumes for sequencing.
! WARNING ! CHEMICAL HAZARD. Formamide is a known
teratogen. It can cause birth defects.Wash thoroughly after handling
formamide.Wear appropriate protective eyewear, clothing, and gloves.
Obtain a copy of the MSDS from the manufacturer.Wash thoroughly after
handling formamide.
Suggested
GeneScan Load
Volumes
The following table lists the suggested load volumes for GeneScan.
Note For more details, refer to the GeneScan Reference Guide and the LMS
v.2 User’s Manual.
No. of Wells Resuspension Vol. (µL)a
a. 5:1 Deionized formamide to 50 mg blue dextran/mL in 25 mM
EDTA
Loading Vol.
(µL)
24/36 6–9 1.5
48 2–4 1.0–1.5
64 2–4 1.0–1.5
96 2–4 1.0–1.5b
b. Loading 1.5 µL requires a syringe with a 0.2-mm tip to facilitate
loading at the bottom of the well.
No. of Wells Loading Vol. (µL)
24/36 1.5
50 1.0–1.5
66 0.5–1.0
96 1.0–1.5a
a. Loading 1.5 µL requires a syringe with a
0.2-mm tip to facilitate loading at the bottom
of the well.
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Gels 2-9
Suggested Load
Mapping The following schematic depicts a microtiter plate showing the
suggested load mapping for an eight-channel loader.
Notes:
♦Loading in staggered format with a two-pitch, eight-channel loader takes 12
loading steps (Load No. 1–12).
♦The number in each well represents the respective gel lane position.
♦Odd-lane loading positions are color coded on the comb.
Gel lane position
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2-10 Gels
Suggested Loading
Sequence Follow the loading procedure to load the odd lanes first, electrophorese
for 2 min, then load the even lanes.
IMPORTANT If any wells leak, flush the contaminated lanes then follow the
table below.
Loading
Procedure To load the gel:
If you are running... Then...
Sequencing Electrophorese immediately, then after each three
loads.
GeneScan Leaking wells are not tolerated in GeneScan
applications.If a well leaks, it is best to run another
gel. At the very least, do not use the wells around
the leaking lane.
Step Action
1Press PAUSE during the prerun.
2Flush the wells with 1x TBE using a syringe.
Note Use care when flushing the wells:Too much pressure
could tear the wells, and touching the teeth with the syringe could
damage the comb and displace the teeth, which could cause
leakage.
3Using the two-pitch, eight-channel loader, draw 2 µL of sample into
the needles.
4Clear any air gaps in the needles by dispensing 0.5 µL of sample,
or by dispensing until sample is visible in the tips of the syringe.
5Using the comb markers as a guide, align the needles into their
respective lanes.
6Very slowly dispense up to 1.5 µL of samples into the wells.Load
the odd lanes first.
Note For longer reads, load the samples close to the gel surface
rather than from the top of the well.To accomplish this, the two-
position loading syringe must have needles with 0.2-mm or 0.25-
mm outer diameters.With 0.3-mm outer diameter needles, the
samples must be gravity loaded.
7After each loading, rinse the needles with warm dH2O and blot dry
with a lint-free tissue to remove residual salt and prevent clogging.
8Continue to load until all odd lanes have been loaded.
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Gels 2-11
9Electrophorese for 2 min.
10 Repeat steps 2–7 to load the even lanes.
11 End the prerun and begin the run.
Step Action
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