Beckman Coulter CEQ 8000 series User manual

TM
390216-AB
July 2003
Beckman Coulter, Inc.
4300 North Harbor Boulevard, Fullerton, CA 92835
Copyright 2003 Beckman Coulter, Inc.
CEQ™ 8000
Series
Genetic
Analysis
System
Sequence Analysis
Troubleshooting
Guide

Copyright, Licenses and Trademarks
 Beckman Coulter, Inc., 2003. All rights reserved. No part of this publication may be
reproduced, transcribed, transmitted, or translated into any language in any form by any means
without the written permission of Beckman Coulter, Inc.
The software is copyrighted and may not be altered or given to a third party without the written
authorization from Beckman Coulter.

Sequence Analysis Troubleshooting Guide iii
Table of Contents
Section I: General Diagnostic Guidelines...............................................................................1
Raw Data - Good Raw Data Signal...............................................................................1
Current Profiles – Normal Current Profile ....................................................................2
Section II: Troubleshooting a CEQ System Problem............................................................4
Is it an instrument problem or a chemistry problem?....................................................4
Diagnostics using CEQ Sequencing Test Sample ..................................................4
Validation of the DTCS kit components.................................................................5
Suspect Customer Supplied Reagents.....................................................................5
Identification of Customer Supplied Template or Primer Problems ......................5
Current problems ...........................................................................................................6
Electrokinetic Injection theory................................................................................6
Diagnosing Current Problems (What does the current profile look like?) .............6
An Erratic current or “Crashed Current” ................................................................7
An Identical Erratic current in all eight Capillaries ..............................................10
Raw Signal Problems ..................................................................................................12
Diagnosing signal level problems.........................................................................12
Causes of Low Raw Data Signal ..........................................................................12
Raw Data Signal that is Too High...............................................................................21
Insertions Caused by n-1 primers .........................................................................22
Insertions Caused by Mixed Templates,
PCR Primer Carryover or Primer Mis-priming ....................................................24
High Baseline Levels ............................................................................................26
Section III: Sequencing Troubleshooting Quick Reference Table .....................................27
Section IV: Sequence Analysis FAQ Sheet............................................................................32
General Operation .......................................................................................................32
Can I perform DNA sequencing and fragment analysis
on the same plate using the CEQ 8000 Series System?........................................32
Can I switch to another database to inspect old data
while new data is being collected?........................................................................32
What are the differences between the LFR-1, LFR-a, LFR-b and
LFR-c methods?....................................................................................................32
Chemistry ....................................................................................................................33
Which primer purification method is best for sequencing primers?.....................33
How do I optimize the Preheat treatment?............................................................35
I have sufficient Raw Data signal but I didn't get a base call.
What can I do? ......................................................................................................36
What can be done to have the greatest chance of accurately
detecting heterozygotes? .............................................................................................37
Appendix A: Technical Bulletins ...........................................................................................44

iv

Section I: General Diagnostic Guidelines
Sequence Analysis Troubleshooting Guide 1
Section I: General Diagnostic Guidelines
The CEQ System has many useful features that allow the user to identify and resolve
problems quickly. The two most useful features in this regard are the Raw Data and
the Current Profile. In certain cases, the analyzed data can be used, but only after the
raw data and current profile have been shown to be good.
RawData-GoodRawDataSignal
The raw data generated by the CEQ System is displayed during the actual run and is
also accessible to the user in the “Analysis Application.”The major diagnostic to use
when looking at raw data is the signal strength. In general, signal strength will
gradually decline as a run progresses as shown below:
In the example shown in Figure 1, with a start time of 20 minutes for the LFR-1
method, the signal strength at the beginning of the run is approximately 70,000 counts,
decreasing to about 10,000 counts at the end of the run. Signal strength is important
because without sufficient signal, it is very unlikely that accurate base calls can be
made. The above example shows excellent signal strength; however, it is not necessary
for each sample to have such high signal in order to obtain accurate base calls. The
important number to remember is signal to noise ratio. With a signal to noise ratio of
about 3:1, the software should give an accurate base call. For example, a profile with a
starting signal strength of 25,000 counts and an ending signal strength of 5,000
(assuming a background of about 1,500) should still give an accurate base call.
Figure 1: Good Raw Data Signal
Profile starts high, gradually decreases in
signal strength as the run progresses

Section I: General Diagnostic Guidelines
2 CEQ™ 8000 Series Genetic Analysis System
Current Profiles –Normal Current Profile
The CEQ System continuously monitors current in all 8 capillaries while the system is
running. This current trace can be extremely useful in diagnosing certain problems.
The current profile should look similar to Figure 2.
The current ramps up to the final level in one stage. The final current level should be
approximately 5–9µA. It is determined by the separation voltage, which is set in the
method and is maintained throughout the run.
Figure 2: Typical LFR-1 Current Profile
N
o current ramp up to the
final separation level in
three stages.
Current remains steady
throughout the separation
time

Section I: General Diagnostic Guidelines
Sequence Analysis Troubleshooting Guide 3
The software allows for the development of custom separation methods. If a
significantly higher separation voltage (6–12KV) is desired, then the advanced
features of assigning the separation conditions should be employed. The advanced
features allow for a gradual, stepped ramp to the final separation voltage. Such a
gradual voltage increase allows the linear polyacrylamide (LPA) to slowly heat up and
thereby reduces possible thermal expansion out of the capillary caused by the rapid
exposure to high voltages. An example of such a three step ramp is given in Figure 3.
Figure 3: Example of a three step ramp
Current ramps up to the
final separation level in
t
hree sta
g
es.
Current remains steady
throughout the rest of the
se
p
aration time

Section II: Troubleshooting a CEQ System Problem
4CEQ™8000 Series Genetic Analysis System
Section II: Troubleshooting a CEQ System Problem
Is it an instrument problem or a chemistry problem?
A systematic approach to troubleshooting should be adopted to effectively diagnose
problems with the CEQ System. The first test that should be done on the CEQ System
is to run the CEQ Sequencing Test Sample. Upon successful completion of CEQ
Sequencing Test Sample analysis, sequencing of the pUC18 control DNA should be
performed. Finally, customer samples should be sequenced. These three tests rely on
the analysis of Raw Data signal, Current profiles and Analyzed Data integrity.
Listed below are more detailed descriptions of the tests in the 3-step diagnostic
process.
Diagnostics using CEQ Sequencing Test Sample
The CEQ Sequencing Test sample is used to confirm the integrity of the instrument
and various reagents used by the system. This test uses a “pre-made”DNA sequencing
reaction prepared using the same pUC18 DNA template as that provided in the CEQ
2000 DTCS kit. The CEQ Sequencing Test Sample should produce data that has a
beginning fluorescence of approximately 50,000–120,000 units and then declines
gradually until the end of the run (viewed in the Raw Data view of the Sequencing
Analysis application). In addition, the current profiles should also be normal (see
section “Current Profiles –Normal Current Profile”on page 2). Finally, the actual base
calls are checked to see if the instrument passes the system specification of 98%
accuracy at 700 bases (if using the LFR-1 method). You should perform the Service
Alignment with the control template pUC18dG. If the CEQ Sequencing Test Sample
provides acceptable results (good raw data signal, normal current profile and meets the
system specifications of 98% accuracy at 700 bases), then the CEQ instrument and
reagents are fine and further diagnostics of the sequencing chemistry are needed. If the
CEQ Sequencing Test Sample does not yield an acceptable result try testing the other
system consumables such as the separation gel, the capillary array and another lot of
the CEQ Sequencing Test Sample. The individual components should be tested in
separate experiments to identify the faulty component. If an acceptable result is still
not achieved after substituting all new components call the Beckman Coulter Field
Service Representative for service on the CEQ system.

Section II: Troubleshooting a CEQ System Problem
Sequence Analysis Troubleshooting Guide 5
Validation of the DTCS kit components
After determining that the CEQ System hardware, gel and capillary are functioning
properly, prepare sequencing reactions with the pUC18 control template and the -47
sequencing primer provided in the DTCS kit. This test is designed to try to
discriminate if the chemistry problems are due to the Beckman Coulter, Inc. provided
reagents or customer supplied reagents. Results that pass the system specification of
98% accuracy at 700 bases (using the LFR-1) confirm the quality of the Beckman
Coulter, Inc. reagents and the customer supplied materials except for primer and
template. If the test does not pass the 98% accuracy at 700 bases (with good raw data
signal and normal current profiles) perform the test again using a new DTCS kit
(preferably of a different lot number). Failure to pass the 98% accuracy with a second
kit would indicate that customer supplied reagents or the post sequencing reaction
cleanup method are suspect. If the cause of the problem is customer supplied reagents
proceed to the next section.
Suspect Customer Supplied Reagents
The customer uses several reagents that may have an impact on the raw data signal and
the current profile. These consist mainly of the chemicals used for the ethanol
precipitation of the finished sequencing reactions such as sodium acetate, EDTA and
ethanol. In some cases with older lots of DTCS kits customer supplied formamide may
also be an issue. The usual symptoms for poor quality sodium acetate, EDTA, ethanol
or formamide are low raw data signal and occasionally erratic current profiles. Using
sodium acetate and EDTA solutions supplied by Sigma as well as the Sample Loading
Solution (SLS) supplied in the DTCS kits will help eliminate problems caused by poor
quality reagents. If one of these reagents is suspect, replace each reagent in a
systematic approach (one at a time) to identify the faulty reagent. If the control
template passes the system specification of 98% accuracy at 700 bases (using the
LFR-1 method) and the Raw Data Signal and Current profiles look good, but the
customers templates do not yield acceptable results proceed to the next section.
Identification of Customer Supplied Template or Primer
Problems
After demonstrating that pUC18 control template and the -47 primer function
correctly, customer DNA templates should be sequenced. If possible, reagents should
be tested individually. This will allow the most efficient identification of problem
reagents. For example, if the customer is using a DNA template that can accommodate
the -47 sequencing primer, then side by side comparisons of sequencing reactions
performed with customer provided primer and the Beckman Coulter, Inc. provided
primer can be conducted.
Using the previously described 3-step process, a CEQ System user should be able to
identify and correct many of the most common problems found in CE based DNA
sequencing. Listed below are several DNA sequencing chemistry problem areas with
their corresponding symptoms and possible corrective actions.

Section II: Troubleshooting a CEQ System Problem
6CEQ™8000 Series Genetic Analysis System
Current problems
Electrokinetic Injection theory
Current problems are associated with the electrokinetic injection of sample and the
subsequent separation of the DNA sequencing fragments in the Linear Polyacrylamide
(LPA) matrix. The electrokinetic injection system will “load”negatively charged
molecules on to the LPA matrix at the tip of the capillary. These negatively charged
molecules include the dye-terminated sequencing fragments which are desirable, and
molecules like the DNA template, proteins, salts, dNTPs and the unincorporated
dye-terminators that are not desirable. Because the CEQ System uses electrokinetic
injection to load DNA sequencing fragments on to the LPA in the capillary, it is
susceptible to “overloading”problems. The most problematic of these molecules is the
DNA template from the DTCS reaction. Listed below are examples of current
problems caused by DNA templates, and to a much lesser extent proteins and other
large, charged macromolecules.
Diagnosing Current Problems (What does the current profile
look like?)
A gradually decreasing current
Supercoiled DNA templates, like plasmid DNA molecules, will load on to the LPA
matrix in the “inlet side”of the capillary and impede the flow of current through the
capillary. When the supercoiled DNA molecules load on to the LPA in the tip of the
capillaries they become “plugged”and do not allow the free flow of electrical current
through the capillary. Plugged capillaries exhibit two different current problems. In the
less severe case where the current profile shows a gradual decline without any erratic
fluctuations in the current, short reads are observed. These short reads are due to the
general slowing of the electrophoretic separation of the DNA fragments causing fewer
dye-labeled DNA fragments to pass through the detection window so fewer bases are
separated. In addition, other macromolecules that co-purify with the DNA template
can also cause current problems. These macromolecules include proteins,
glycoproteins, bacterial chromosomal DNA, RNA and cell wall debris. In general,
linear DNA molecules such as PCR products or restriction endonuclease digestion
products do not cause current problems.
Corrective actions to solve minor current problems
A gradual decrease in the current throughout the run indicates that there may only be a
slight excess of the template DNA in the sequencing reaction or the template is very
highly supercoiled. There are several solutions to this problem (listed from simplest to
most elaborate):
1. If sufficient raw data signal is present, often just reducing the injection time is
adequate to improve the sequence call and read length. Simply rerunning the
sample and injecting for half the time may solve the problem (i.e. if the first
injection was 2kV for 15 seconds, use 2kV for 7.5 seconds).

Section II: Troubleshooting a CEQ System Problem
Sequence Analysis Troubleshooting Guide 7
2. If sufficient raw data signal is present, reduce the amount of template present in
the cycle sequencing reaction. In this case, just adding half the amount of
template may eliminate the current problem (i.e. if the original template amount
was 100 femtomoles (fmoles), use 50 fmoles).
3. Use the preheat treatment of the template DNA prior to the cycle sequencing
reaction. The preheat treatment (see CEQ DTCS Chemistry Guide, Beckman
Coulter, Inc. P/N 718119), nicks the template DNA which alleviates the
supercoiling of the plasmid DNA. As noted above, supercoiled DNA templates
are the primary cause of current problems.
4. Use combinations of 1, 2 and 3. Using various combinations of the first three
recommendations may also eliminate current problems. For example, use the
preheat treatment on 50 fmoles of template.
5. Use a DNA template purification process that eliminates macromolecules such
as proteins, glycoproteins, bacterial chromosomal DNA, RNA and cell wall
debris. Most commercial DNA preparation methods yield template DNA that is
essentially free of contaminating macromolecules.
6. Remove the template from the sequencing reaction. This is a very elaborate
process requiring special primers and reagents, but also yields sequencing
reactions that produce no current problems. The most common technique is to
use a biotinylated sequencing primer and the DynaPureII™sequencing reaction
cleanup kit (Dynal, Inc. Oslo, Norway, product number 603.05) to purify
sequencing reaction products from contaminants such as the template DNA,
unincorporated dye-terminators, dNTPs and other salts.
An Erratic current or “Crashed Current”
In the more severe case where the current profile is erratic, the passage of dye-labeled
DNA fragments through the detection window does not pass at a constant rate and
sequencing errors are introduced. The sequencing software expects the fluorescent
molecules to be detected at a fairly constant rate, this makes predictions of the base
spacing in the base calling process. If the peak spacing is “expanded”(too few bases
per minute) as found in regions were the current has dropped quickly, the software
may insert extra bases into the sequence. Shown below are examples of erratic current
and crashed current, respectively.

Section II: Troubleshooting a CEQ System Problem
8CEQ™8000 Series Genetic Analysis System
If the current is unstable or erratic, then the migration times of the bases will be
adversely affected and this can lead to inaccuracies in the base calling. During the time
that the current is not being maintained, as shown in Figure 4, the base migration will
be slowed and the accuracy of base calling will be low. In addition to inaccuracies in
the base call the total read length will also be diminished simply due to the fact that the
migration of the bases has been slowed from the normal run conditions.
In other cases, current failures can be more significant. Figure 5 shows a current
profile that fails to reach the final running current after the normal ramping. Note that
the current never reaches the normal running current of 5–9µAmpsandalsodeclines
rapidlytolessthan2µAmps.
Figure 4: Erratic Current
Figure 5: Crashed Current
Current ramps normally
but then dips down and
recovers.
Does not achieve normal current level
Rapid decline in the current
Low separating current

Section II: Troubleshooting a CEQ System Problem
Sequence Analysis Troubleshooting Guide 9
Corrective actions to solve erratic current or current crashes
The occurrence of erratic current profiles indicates that there is a more severe DNA
template-overloading problem. In this case more drastic measures may need to be
taken. In the situation were erratic current is observed, simply rerunning the sample
and injecting for half the time will not solve the problem. Listed below are some of the
steps the should be made to eliminate the erratic current problem:
1. If sufficient raw data signal is present, reducing the amount of template present
in the cycle sequencing reaction is an obvious first step. In this case, adding half
the amount of template may eliminate the current problem (i.e. if the original
template amount was 100 fmoles, use 50 fmoles).
2. Use of the preheat treatment of the template DNA prior to the cycle sequencing
reaction is highly recommended for supercoiled plasmid samples and is required
to solve erratic current problems or current crashes. Once again, the preheat
treatment (see CEQ DTCS Chemistry, Beckman Coulter, Inc. P/N 718119) nicks
the template DNA to alleviate the supercoiling of the plasmid DNA and nicked
DNA does not “clog”the capillaries during injection. In many cases the preheat
treatment of plasmid templates at 96°C for 1 minute prior to adding the template
to the sequencing reaction can eliminate current irregularities (see Figure 6).
Figure 6: Preheat treatment of plasmid templates

Section II: Troubleshooting a CEQ System Problem
10 CEQ™8000 Series Genetic Analysis System
3. An alternative to preheat treatment is to cleave the plasmid with a restriction
endonuclease before adding the template to the sequencing reaction. In effect,
this method converts a supercoiled plasmid into a linear molecule, which causes
the DNA template to perform like a PCR product (no current problems
observed). This method, although very effective, requires some knowledge of the
DNA sequence to know where to “cut”the plasmid DNA. The plasmid needs to
be cut at a position that is 5' to the sequence that is desired. For example if the
primer binds to the plasmid at position 100 and sequence from position 100 to
600 is desired then the restriction endonuclease recognition site should be at
position 601 or greater.
4. Removing the template from the sequencing reaction is the only guaranteed
method for curing the current instability problem. This is a very elaborate
process requiring special primers and reagents, but also yields sequencing
reactions that produce no current problems. The most common technique is to
use a biotinylated sequencing primer and the DynaPureII™sequencing reaction
cleanup kit (Dynal, Inc., Oslo, Norway, product number 603.05). This purifies
the sequencing reaction products from contaminants such as the template DNA,
unincorporated dye-terminators, dNTPs and other salts.
An Identical Erratic current in all eight Capillaries
If all eight capillaries have identical erratic current problems, then the cause of the
problem is a block in the electrical path on the “manifold side”of the CEQ gel system.
A bubble forming in the electrode chamber which blocks the flow of current through
the system most often causes this problem. The result of the blocked current flow is
that all eight capillaries will have reduced read lengths and probable base calling
errors. This is also an indication that there is a system problem and not a problem with
the individual samples. The bubbles may be caused by a variety of reasons: excessive
removal and installation of gel cartridges, improper sealing of the manifold plug of the
capillary array in the gel manifold gasket, a cracked electrode housing, etc.

Section II: Troubleshooting a CEQ System Problem
Sequence Analysis Troubleshooting Guide 11
Shown in Figure 7 are the current profiles of a set of capillaries with an air bubble on
the manifold side of the CEQ gel system.
Corrective actions to solve manifold bubble current problems
1. Do 1–2 manifold purges from the Direct Control menu on the CEQ Run
application.
2. Check the sample plate for air bubbles before loading it onto the CEQ.
3. Call Beckman Coulter, Inc. service if the first two actions do not fix the problem.
Figure 7: Manifold bubble current problem

Section II: Troubleshooting a CEQ System Problem
12 CEQ™8000 Series Genetic Analysis System
RawSignalProblems
Types of Raw data signal problems
There are several types of raw data signal problems. The most common problem is
signal levels that are too low. In this case there is insufficient fluorescence signal
(shown as Activity (cnts) on the electropherogram) to generate an accurate base call.
Another possible problem is when there is too much raw data signal, which causes the
photomultiplier detection system to be saturated. This is caused by too many
fluorescently labeled DNA sequencing fragments. Another category of raw data signal
problem is too many fluorescent peaks in the electropherogram. This can be caused by
two independent sequencing events present in the same sample. This phenomenon is
caused by either having two or more templates in the reaction or multiple (2 or more)
primer binding sites on a DNA template. A variation on the previous problem is
referred to as a “pre-peak problem”. Prepeaks are caused by poor quality primer
synthesis where a high proportion of n-1 primer (primers which are one base short of
the desired length) are present. The previously mentioned problems, as well as several
other possible problems, will be discussed in more detail in the following sections.
Diagnosing signal level problems
The first step in diagnosing a Raw Data signal problem is to look at the fluorescent
signal (Raw Data signal) in the Sequencing Analysis application. The data, which is
plotted as Activity (cnts) versus Time (Minutes), should show fluorescent signal
(Activity cnts) in the range of 5,000–125,000 cnts. The signal will be high in the
beginning and gradually decline throughout the run.
Causes of Low Raw Data Signal
Low Raw Data Signal due to not enough template
Low raw data signal can be caused by a variety of issues. One of the most common
causes is lack of sufficient DNA template in the cycle sequencing reaction. It is vital to
the success of CE sequencing to have the correct amount of template in the reaction. It
is recommended that 50–100 fmoles of plasmid DNA be used in the cycle sequencing
reaction. This provides enough template to generate an adequate amount of
fluorescently labeled DNA sequencing fragments yet not so much as to cause current
problems (see “Current problems”on page 6). Half this amount of DNA template
(25–50 fmoles) should be used for single stranded DNA templates such as M13 phage
DNA and even less DNA is needed for small PCR products (10–50 fmoles for PCR
products less than 3KB in length). In many cases the amount of template added to the
reaction is not determined and therefore, insufficient template is present. In other
cases, an incorrect approximation of the DNA concentration is made.
Spectrophotometric estimation of DNA samples is only valid if the DNA is pure (as in
the case of commercial DNA template purification methods). Crude preparation of
DNA templates which have substantial amounts of protein and/or RNA will over
estimate the concentration of the template and cause the user to add too little DNA to
the sequencing reaction (as in the case of crude alkaline lysis minipreps).

Section II: Troubleshooting a CEQ System Problem
Sequence Analysis Troubleshooting Guide 13
Shown, in Figure 8, is an example of a DNA sequencing sample where not enough
template DNA was added. In this example less than 25 fmoles of template was added
to the sequencing reaction. Notice the very low Raw Data signal throughout the run
and particularly the low Raw Data signal in the red, green and black traces. An easily
identifiable indicator of low template concentration and, in general, a poor sequencing
reaction is a high amount of unincorporated dye-terminators (note the large peaks at
around 40 minutes).
Corrective actions
1. Add the correct amount of the template DNA to the reaction. This will require
quantitation of the template DNA by spectrophotometry (in the case of
commercial DNA preparations) or by estimation using agarose gel
electrophoresis and comparison to a know quantity of DNA.
2. Alternatively, the user could try a dilution series with the same template starting
with an amount that is obviously too high and ending with an amount which is
much too low. This method assumes that the user knows the approximate amount
of template added to the reaction (this may be from previous work using similar
DNA preparation methods).
3. Use the preheat treatment for highly supercoiled plasmids. Most commercial
DNA preparations yield highly supercoiled plasmids. The preheat treatment will
knick the supercoiled plasmid which yields much more efficient DNA
sequencing (linear molecules sequence better than supercoiled molecules).
Figure 8: Low Template amount
Low signal
Unincorporated
Dye-Terminators

Section II: Troubleshooting a CEQ System Problem
14 CEQ™8000 Series Genetic Analysis System
4. If the correct amount of template was added and the preheat treatment does not
yield a substantial increase in Raw Data signal increase the number of cycles in
the thermal cycling program from 30 to 40 or 50.
5. If the correct amount of template was added and the Preheat Treatment and / or
increasing cycle number does not yield a substantial increase in Raw Data signal,
a DNA Polymerase inhibitor may be present (do not resuspend DNA in DEPC
treated water). In this situation further purification of the DNA template may be
required. In some cases a simple ethanol precipitation of the plasmid will remove
the inhibitor, whereas other situations may require the use of commercial DNA
preparation methods such as the Qiagen QiaQuick kit.
Low Raw Data Signal Due to Primer Quantity and Quality Problems
The amount and purity of sequencing primers can also cause low raw data signal. For
example, having insufficient quantities of the primer or having a poor quality primer
(e.g. a primer that has not been totally deprotected after synthesis) will yield low Raw
Data signal. Many suppliers of oligonucleotides provide sufficiently pure preparations
of primers. Most of these vendors will provide “desalted primers”which function well
for DNA sequencing. However, further purification of the primers by TSP (Trityl
Specific Purification) or HPLC (High Performance Liquid Chromatography) is highly
recommended when the highest quality DNA sequencing results are desired. Also,
primers purified by denaturing gel electrophoresis have been shown to contain
inhibitors of the DNA Polymerase and therefore, are not recommend for use in DNA
sequencing reactions.
Primer Design Problems that Cause Low Raw Data Signal
Another cause of primer related low raw data signal is poor primer design. Primers
should be designed so that they have reasonably high Tm’s(T
mgreater than 55°C), no
internal secondary structures (such as hairpin loops) and a low probability of forming
“primer dimers”(the 3' ends of the primers binding to each other and are extended).
The Tmoftheprimermustbeatleast5°C higher than the annealing temperature of the
cycle sequencing program (50°C) to adequately bind to the template DNA and be
extended by the polymerase. Internal secondary structures and primer dimers are
thermodynamically favored over primer- template binding so they reduce the effective
concentration of the primer in the sequencing reaction.

Section II: Troubleshooting a CEQ System Problem
Sequence Analysis Troubleshooting Guide 15
In the following examples (Figure 9 and Figure 10), the template is the same, but two
different primers have been used. Primer 1 has a Tmof 43.5°C, a 31.6% GC content
and is 19 bases long. Primer 2 has a Tmof 79.8°C, a 62.5% GC content and is 24 bases
long. The effect on the data obtained from the CEQ is clear. With a less than efficient
reaction, the signal level is low and the base calling accuracy is not optimal.
Figure 9: Primer 1
Figure 10: Primer 2

Section II: Troubleshooting a CEQ System Problem
16 CEQ™8000 Series Genetic Analysis System
Corrective Actions
1. Add the correct amount of the primer to the reaction. This will require
quantitation of the primer by spectrophotometry. The recommended amount of
primer is at least 40-fold molar excess in relation to the template. For sequencing
of most template DNAs, 3–10 pmoles of primer is sufficient for a successful
sequencing reaction.
2. Make sure that the primer preparation is pure. Using reputable oligonucleotide
vendors is your best way to get high quality primers. If possible have the primers
purified by TSP or HPLC.
3. Always design the sequencing primers for use in Cycle sequencing reactions.
This means designing primers with high Tm’s and no possibility of forming
secondary structures or primer dimers. There are many DNA analysis programs
available for checking primer design.
4. Make sure that the primer is resuspended in a solution that has no EDTA or
DEPC present. Molecular biology grade water or 10mM Tris pH 8.0 are good
resuspension solutions.
5. Increase the number of cycles in the thermal cycling program from 30 to 40 or
50.