Thermo Fisher Scientific ABI PRISM® 310 Genetic Analyzer: A Fast Native Protocol Owner's manual

Brand: Thermo Fisher Scientific

Model: ABI PRISM® 310 Genetic Analyzer: A Fast Native Protocol

Type: Owner's manual

User Bulletin

ABI P

RISM

310 Genetic Analyzer

June 23, 1999 (updated 12/2000)

A Fast Native Protocol for the Analysis of PCR Products

In This Bulletin

This User Bulletin describes the Fast Native Protocol for the separation of DNA

fragments that are

500 bp in length.

The following topics are covered in this User Bulletin:

What is the Fast

Native Protocol?

The Fast Native Protocol is a rapid capillary electrophoresis protocol for separating

DNA fragments that are

500 bp in length. The Fast Native Protocol is run on the

ABI P

RISM®

310 Genetic Analyzer using a shortened (41-cm) capillary and 7-minute

analysis times.The sizing resolution is at least 5 bp.

Advantages

The advantages of the Fast Native Protocol are as follows:

♦

Separates DNA fragments using 7-minute analysis times

♦

Processes 96 samples in less than 12 hours

♦

Assesses the performance of speciﬁc reactions

– PCR, for sizing of fragments that differ by at least 5 bp

– Minisequencing

– Detection of the absence or presence of DNA fragments in a sample

Limitations

The Fast Native Protocol has only been shown to separate DNA fragments that are

500 bp in length.

Topic See Page

Safety 2

Before You Begin 3

Material Preparation 4

Preparing for a Run 6

Analyzing the Data 9

Technical Support 12

Page 2 of 16 ABI P

RISM

® 310 Genetic Analyzer

Safety

Documentation User

Attention Words

Five user attention words appear in the text of all Applied Biosystems user

documentation. Each word implies a particular level of observation or action as

follows.

Note

This word is used to call attention to information.

IMPORTANT

This word calls attention to information that is necessary for correct operation of

the kit or instrument.

CAUTION

This word informs the user that damage to the instrument could occur if the

user does not comply with the information. It also indicates a potentially hazardous

situation that could result in minor or moderate injury to the user.

! WARNING !

This word informs the user that serious physical injury to the user or

other persons could occur if these required precautions are not taken.

DANGER

Indicates an imminently hazardous situation that, if not avoided, will result in death

or serious injury

General Warning

! WARNING !

CHEMICAL HAZARD. Some chemicals used with Applied Biosystems

instruments or kits may be hazardous and can cause injury, illness, or death. Check

regularly for chemical leaks and chemical spills, which represent potential safety

hazards to the operator. If a leak or a spill occurs, follow clean-up instructions in the

Material Safety Data Sheets (MSDSs), or in the Waste Proﬁle in the Site Preparation and

Safety Guide. Hazardous Chemical Warnings are prominently displayed on the labels of

all hazardous materials. Always read the appropriate MSDSs and Waste Proﬁles before

using the chemicals in any way.

User Safety

Precautions

Always wear the appropriate protective gloves, clothing, and eyewear when handling

chemicals.

Ordering MSDSs

Material Safety Data Sheets (MSDSs) for hazardous chemicals manufactured by

Applied Biosystems will accompany your ﬁrst shipment. To receive additional copies of

MSDSs at no extra cost, call Applied Biosystems at (800) 327-3002.

For chemicals required for this instrument but not manufactured or sold by Applied

Biosystems, please obtain the MSDSs from their manufacturers.

Ordering Kits and

Reagents

To order ABI P

RISM®

kits or reagents, please contact Applied Biosystems at one of the

regional sales ofﬁces listed in the Technical Support section at the end of this User

Bulletin. Please have the part number of the kit or reagent you are ordering available

when ordering.

ABI P

RISM

® 310 Genetic Analyzer Page 3 of 16

Before You Begin

Materials Required

You will need the following materials to perform a Fast Native Protocol run:

Equipment/Reagent Item Source

ABI P

RISM

310 Genetic Analyzer

See your local Applied Biosystems

representative

ABI P

RISM

310 Data Collection Software, version

1.2 or higher

GeneScan

Analysis Software for the 310 Genetic

Analyzer, version 2.0.2 or higher

Glass syringe, 1.0-mL Applied Biosystems

(P/N 4304471)

GeneScan

capillary, 47-cm x 50-

m i.d. (labeled

with a green mark)

Applied Biosystems

(P/N 401823)

Genetic Analyzer capillary cutters Applied Biosystems

(P/N 401958)

310 GeneScan

Polymer (7%) Applied Biosystems

(P/N 401885)

310 Genetic Analyzer Buffer with EDTA Applied Biosystems

(P/N 402824)

Dye Primer Matrix Standards Kit

Fluorescent Amidite Matrix Standards Kit

plus NED Matrix Standard

Applied Biosystems

(P/N 401114)

Applied Biosystems

(P/N 401546)

(P/N 402996)

GeneScan

-350 Internal Lane Size Standard

GeneScan

-500 Internal Lane Size Standard

Applied Biosystems

(P/N 401735)

(P/N 401734)

Genetic Analyzer Buffer Vials (4.0-mL) Applied Biosystems

(P/N 401955)

ABI P

RISM

310 Genetic Analyzer User’s Manual

(P/N 903565)

GeneScan Analysis Software User’s Manual

(P/N 904435)

GeneScan Reference Guide

(P/N 4303189)

1.5-mL Eppendorf tube with the lid removed Major Laboratory Suppliers (MLS)

Magnifying glass MLS

Metric ruler MLS

Heat block MLS

Deionized formamide

Note

Use high-quality deionized formamide.

MLS

Page 4 of 16 ABI P

RISM

® 310 Genetic Analyzer

Material Preparation

Shortening the

Capillary

Preparing Stock

Solutions

Prepare stocks of the following reagents/buffers to save time during run setup:

♦

Fast Native Protocol buffer (0.6X 310 Genetic Analyzer Buffer with EDTA)

♦

Fast Native Protocol polymer (2% 310 GeneScan Polymer solution in 0.6X 310

Genetic Analyzer Buffer with EDTA)

Note

Use the Fast Native Protocol buffer and polymer in place of the standard solutions when

running samples.

Making the Fast Native Protocol Buffer

Note

The Fast Native Protocol buffer lasts for 4 weeks at 2–6 °C. Warm the buffer to room

temperature before use.

To cut the capillary:

Step Action

Have one of each at hand:

♦

GeneScan capillary (green)

♦

Capillary cutter

♦

Metric ruler

♦

Magnifying glass

Align the 41-cm end of the capillary on top of the metric ruler.

Using the capillary cutter, score the capillary at 6 cm from the end farthest from the

capillary window.

Break off the capillary at the score. The total capillary length is now 41 cm.

Note

Properly dispose of the 6-cm piece of the capillary.

Check for a clean cut at the tip with a magnifying glass.

If the cut does not appear clean, smooth the end by lightly brushing the ﬂat end of

the capillary cutter over the end of the capillary.

To prepare 50 mL of Fast Native Protocol buffer:

Step Action

To a 50-mL polypropylene tube add:

♦

3.0 mL 310 Genetic Analyzer Buffer with EDTA

♦

47 mL deionized water

Mix thoroughly.

ABI P

RISM

® 310 Genetic Analyzer Page 5 of 16

Making the Fast Native Protocol Polymer

Note

Fast Native Protocol polymer lasts for 4 weeks at room temperature.

To prepare 50 mL of Fast Native Protocol polymer:

Step Action

To a 50-mL polypropylene tube add:

♦

14.3 mL 310 GeneScan Polymer (7%)

♦

3.0 mL 310 Genetic Analyzer Buffer with EDTA

♦

32.7 mL deionized water

Mix thoroughly.

Page 6 of 16 ABI P

RISM

® 310 Genetic Analyzer

Preparing for a Run

Instrument Setup

Refer to the

ABI P

RISM

310 Genetic Analyzer User’s Manual

for the general

procedure.

Setting the Run

Temperature

Set the instrument run temperature to 60 °C immediately before run setup. Refer to

the

ABI P

RISM

310 Genetic Analyzer User’s Manual

for details.

At Any Time Before

a Run

Perform the following at any time before beginning a run:

♦

Complete the Sample Sheet.

Refer to the

ABI P

RISM

GeneScan

Analysis Software Manual

for details.

♦

Create an appropriate Fast Native Protocol matrix ﬁle for the chosen dye set.

For directions on preparing matrix samples for Fast Native Protocol analysis, see

“Preparing the Matrix Samples” on page 7.

♦

Create and then reuse a single matrix ﬁle for each set of run conditions.

♦

For DNA fragments that are considerably smaller than 500 bp, determine in a trial

run the approximate electrophoresis time in seconds. Then replace the default run

time with this sample-speciﬁc run time.

Preparing and

Loading Samples

To prepare and load the samples:

Step Action

Label the sample tubes with a permanent marker.

To each tube add:

♦

10.5

L deionized formamide

♦

L GeneScan Internal Lane Size Standard (GeneScan-350 or GeneScan-500

recommended)

♦

L sample

Note

When analyzing DNA fragments that are considerably smaller than 500 bp,

determine in a trial run the approximate electrophoresis time in seconds. Then

replace the default run time with this sample-speciﬁc run time.

! WARNING ! CHEMICAL HAZARD. Formamide is a known teratogen

(i.e., 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.

3Place the samples in a sample tray.

4Place the samples in a heat block or thermal cycler for 5 minutes at 95 °C.

5Remove the samples from the heat source and place immediately in an ice-water

bath.

6Place the sample tray on the autosampler.

Note The 96-well tray used in the GeneAmp® PCR System 9700 requires a tray

adaptor (P/N 4305051) to be used with the ABI PRISM 310 autosampler.

ABI PRISM ® 310 Genetic Analyzer Page 7 of 16

(ll

Preparing the

Matrix Samples

To prepare the matrix samples:

Run Modules The Fast Native Protocol has two dedicated modules: Fast GS Template A and Fast

GS Template D.

Starting the Run

Step Action

1To each of four sample tubes add:

♦11 µL deionized formamide

♦1 µL each of one of the matrix standards

! WARNING ! CHEMICAL HAZARD. Formamide is a known teratogen

(i.e., 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.

2Place the standards in a heat block or thermal cycler for 5 minutes at 95 ∞C.

3Remove the standards from the heat source and place immediately in an ice-water

bath.

4Run these four injections either alone or in the ﬁrst four injections of an

experimental run.

The default values for the Fast Native Protocol modules are:

Module Value

Run Time 300 seconds

Electrophoresis Voltage 15 kV

Electrophoresis Temperature 60 °C

Injection Time 5 seconds

Injection Voltage 15 kV

To start the run:

Step Action

1Install a new, 41-cm capillary as described in “Shortening the Capillary” on page 4.

2Select Change Capillary from the Instrument window. Then click OK in the Reset

window to reset the injection counter.

3Set up the Sample Sheet as described in the ABI PRISM 310 Genetic Analyzer

User’s Manual.

Note Prepare the Sample Sheet at any time prior to the run and save it to the

Sample Sheet folder.

4Select New from the File pull-down menu and click the GeneScan Injection List icon.

Page 8 of 16 ABI PRISM ® 310 Genetic Analyzer

5Set up the Injection List as described in the ABI PRISM 310 Genetic Analyzer User’s

Manual.

Be sure to do the following:

♦Select the appropriate Sample Sheet from the Sample Sheet pop-up menu.

♦Select module Fast GS Template A or Fast GS Template D from the Module pop-up

menu for every injection.

IMPORTANT The displayed value in Run Time is in seconds, not minutes. To

change the default value, calculate accordingly.

6Either:

♦Select the appropriate matrix ﬁle from the Matrix pop-up menu for every injection.

♦Start the run and assign the matrix to the sample ﬁles later.

Note If you have not already created a matrix ﬁle, see “Preparing the Matrix

Samples” on page 7. Refer to Appendix B of the GeneScan Reference Guide for

instruction on creating a matrix ﬁle.

7Click the Start button.

Note If you have not preheated the heat plate, the module has an initial step in

which the plate is heated to the selected run temperature before the ﬁrst sample is

injected.

To start the run: (continued)

Step Action

ABI PRISM ® 310 Genetic Analyzer Page 9 of 16

Analyzing the Data

Creating a Matrix

File

You must create a matrix ﬁle before analyzing the Fast Native Protocol data for the

ﬁrst time. For more information on creating matrices, refer to Appendix B of the

GeneScan Reference Guide.

For directions on preparing matrix samples for Fast Native Protocol analysis, see

“Preparing the Matrix Samples” on page 7. Be sure to denature the matrix samples

before loading.

To create the matrix ﬁle, select the matrix standards and module as shown below.

Note Usually, you create and save a single matrix ﬁle for each set of run conditions. However,

if you experience persistent problems, such as spectral overlap in the analyzed data, you

should remake the matrix ﬁle even if you have not altered the run conditions.

If using... Use module... And name it...

Dye Primer Matrix

Standards Kit

Fast GS Template A Fast PCR Matrix A

Fluorescent Amidite Matrix

Standards Kit

plus NED Matrix Standard

Fast GS Template D Fast PCR Matrix D

Page 10 of 16 ABI PRISM ® 310 Genetic Analyzer

Setting the Analysis

Parameters

IMPORTANT If you choose to use different analysis parameters from those recommended

here, be sure to deﬁne the Analysis Range to exclude the primer peak.

Deﬁning the Size

Standard

Deﬁne the size standard so that each unknown peak is ﬂanked by at least two size

standard peaks on either side.

Refer to the GeneScan Analysis Software User’s Manual for detailed protocols.

To set the analysis parameters:

Step Action

1Open the GeneScan Analysis Software (version 2.0.2 or higher).

2Choose New from the File menu and click the Analysis Parameters icon.

3Specify the settings as shown below.

Note When analyzing PCR products, in Analysis Range choose a value for the

start position that begins just after the primer peak.

4Choose Save As... from the File menu.

5Save the settings as Fast Native Analysis Parameters.

6Click OK when done.

ABI PRISM ® 310 Genetic Analyzer Page 11 of 16

Internal Size

Standard

Shown below is the electropherogram for the GeneScan-500 internal lane size

standard, labeled with ROX. Peaks representing 100, 200, 300, 400, and 500 bp are

shaded. The table indicates the electrophoresis time (in minutes) for these peaks.

Note When assigning the size values to the internal size standard peaks, do not assign a value to the 250-bp peak, since

this fragment shows an anomalous migration behavior under the Fast Native Protocol conditions.

Page 12 of 16 ABI PRISM ® 310 Genetic Analyzer

Technical Support

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You can contact Applied Biosystems for technical support by telephone or fax, by

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To Contact Technical

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Contact technical support by e-mail for help in the following product areas:

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877 and Catalyst 800 instruments)

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then press 24

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ABI PRISM ® 310 Genetic Analyzer Page 13 of 16

Outside North America

ABI PRISM® 3100 Genetic Analyzer 1-800-831-6844,

then press 26

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BioMerge™, and SQL GT™

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Reader

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then press 16

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Product or

Product Area

Telephone

Dial...

Fax

Dial...

Page 14 of 16 ABI PRISM ® 310 Genetic Analyzer

Hong Kong 852 2756 6928 852 2756 6968

Korea (Seoul) 82 2 593 6470/6471 82 2 593 6472

Malaysia (Petaling Jaya) 60 3 758 8268 60 3 754 9043

Singapore 65 896 2168 65 896 2147

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Thailand (Bangkok) 66 2 719 6405 66 2 319 9788

Europe

Austria (Wien) 43 (0)1 867 35 75 0 43 (0)1 867 35 75 11

Belgium 32 (0)2 712 5555 32 (0)2 712 5516

Czech Republic and Slovakia (Praha) 420 2 61 222 164 420 2 61 222 168

Denmark (Naerum) 45 45 58 60 00 45 45 58 60 01

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All other countries not listed

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ABI PRISM ® 310 Genetic Analyzer Page 15 of 16

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For Research Use Only. Not for use in diagnostic procedures.

Applied Biosystems, ABI PRISM, and its design are registered trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other

countries. ABI is a trademark of Applera Corporation or its subsidiaries in the U.S. and certain other countries.

GeneScan and GeneAmp are registered trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries.

All other trademarks are the sole property of their respective owners.

P/N 4312955B, Stock No. 106UB07-02

Thermo Fisher Scientific ABI PRISM® 310 Genetic Analyzer: A Fast Native Protocol Owner's manual

Thermo Fisher Scientific ABI PRISM® 310 Genetic Analyzer: A Fast Native Protocol Owner's manual

Thermo Fisher Scientific ABI PRISM® 310 Genetic Analyzer: A Fast Native Protocol Owner's manual

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