User Bulletin
ABI P
RISM®
377 DNA Sequencers
March 25, 1999 (Updated 05/2001)
SUBJECT: Aliquoting Bulk Quantities of ABI P
RISM
®
Cycle
Sequencing Ready Reaction Mixes
Overview
Use the following instructions to aliquot the Ready Reaction Mix from
the 5000-reaction ABI P
RISM®
Cycle Sequencing Ready Reaction Kit.
To maximize ease of handling and to minimize the number of freeze-
thaw cycles, split your Ready Reaction Mix into smaller aliquots upon
receipt. An appropriate volume aliquot receives less than five
freeze-thaw cycles, and contains sufficient quantity for one-week’s
worth of reactions.
Page 2 of 4 User Bulletin: ABI P
RISM®
377 DNA Sequencers
Aliquot
Preparation
To prepare aliquots:
Step Action
1
Estimate the number of cycle-sequencing reactions your laboratory
performs in a week. This determines:
♦
The number of reactions per aliquot
♦
The required tube size
2
Use thick-walled presterilized polypropylene microcentrifuge tubes
or autoclave nonsterile tubes at 121 °C for 20 minutes.
3
Label each tube with the name of the kit, the part number, the lot
number, and the date.
4
Thaw the Ready Reaction Mix on ice or at room temperature,
protected from light. Do not use heat to thaw.
5
Gently and thoroughly mix the Ready Reaction Mix by inverting the
tube several times (about 10–20 seconds) to create a
homogeneous solution. Do not vortex the tube.
IMPORTANT
Storage of diluted Ready Reaction Mix is not
recommended.
IMPORTANT
Wear appropriate eyewear, clothing, and gloves
when working with reagents.
Note
If you plan to dilute the Ready Reaction Mix with 5X
Sequencing Buffer (P/N 4305605 - 600 1/2 reactions; P/N 4305603
- 5400 1/2 reactions) do so just before use. See the Dilution of
BigDye™ Terminators Application Note (P/N 230403).
Number of
Reactions
per Aliquot
Volume (
µ
l)
of Mix per
Reaction
Total
Volume (
µ
l)
Tube Size
(ml)
100 4 400 0.5
8 800 1.5–2.0
200 4 800 1.5–2.0
8 1600 2.0
250 4 1000 1.5–2.0
400 4 1600 2.0
User Bulletin: ABI P
RISM®
377 DNA Sequencers Page 3 of 4
6
Collect the Ready Reaction Mix in the bottom of the tube by flash
centrifugation (approximately 1000 x
g
for 30 seconds.
IMPORTANT
If you experience tube-to-tube variation in sequence
data quality, the reagents may not have been mixed adequately
before aliquotting. If this occurs, pool the separate aliquots, remix,
and redispense.
7
Aliquot the Ready Reaction Mix, capping each tube after it is filled.
Note
Use a positive displacement pipettor or use pipette tips with
a hydrophobic barrier as it is critical not to contaminate the Ready
Reaction Mix with DNA from any source.
Note
Use aseptic technique, with gloves and sterile pipette tips
throughout.
8
Store the tubes upright, shielded from light, in a –20 °C constant
temperature freezer.
IMPORTANT
Do not use a frost-free freezer.
To prepare aliquots:
(continued)
Step Action
© Copyright 2001, Applied Biosystems. All rights reserved.
ABI P
RISM
and its design, and Applied Biosystems are registered trademarks of Applera Corporation or its subsidiaries in the U.S.
and certain other countries.
BigDye is a trademark 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 4310588B
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