A-to-Z SAMPLE PREP FOR DNA SEQUENCING
ON BECKMAN-COULTER CEQ 2000XL
(February 3, 2003)
(To be used in conjunction with protocol supplied with Quick Start Kit)
(A) PRIMER PREPARATION
1-Primer Design:
-The goal here is to maximize priming efficiency and specificity. This in turn will maximize the amount of reaction product from a
specific location on the template. The result will be longer, more accurate base calls. Remember that the longer the primer the less
likelihood of multiple sequence sites.
-The primer which comes with the Quick Start Kit (M13 -47) is a universal cloning plasmid primer with a Tm of 80*C (24 bases,
62.5% GC) and is one possible choice. The sequence of this primer is:
5’-CGC-CAG-GGT-TTT-CCC-AGT-CAC-GAC-3’
It works well with the standard pUC18 template that also comes with the Kit, and with lacZ-containing plasmids pGEM and
pBluescript, but not necessarily with all plasmids used in eukaryotic expression.
-The standard T3 and T7 primers have low Tm’s and therefore don’t work well unless the cycle sequencing program is modified by
lowering the annealing temperature to 45*C. The variations Bluescript T3 and Bluescript T7 should work nicely without modifying
the program because they have much higher Tm’s.
-When designing your own primers remember that there are three characteristics needed to get high efficiency and high specificity:
First, the Tm should be at least 60*C. Second, the GC content should be at least 50%. Third, the length should be at least 20 to 24
bases long. (Assuming that the Tm is roughly 2* per A or T and 4* per G or C, a 20mer with 10 A’s or T’s and 10 G’s or C’s wo uld
have a Tm of about 60*C. A lower GC content or a shorter length might give a Tm that is too low.)
-Primers should not self-anneal (form internal secondary structures) or hybridize within themselves (form “primer dimers”) because
this will reduce priming efficiency.
-For primer design Beckman suggests software Oligo 5.0 (NBI/Genovus) or Oligo 6.0 (Molecular Biology Insight Inc at 800-7474362).
2-Primer Purification:
-Primers should be desalted. (Note that the CEQ capillary machine will tolerate excess primer in the sample but not excess salt. This
is the opposite of the characteristics of the old ABI slab-gel machine.)
-If you are having trouble getting good sequence you might get better results by purifying with “trityl-on” HPLC or OCP columns (but
not acrylamide gel).
3-Primer Amount:
-Beckman suggests that when you are preparing your own primers you should prepare 5uMolar stock solutions and use at least 1ul
per reaction which is 5pmol. If you are using the 1.6uMolar M13 -47 primer that comes with the Kit you need to use 3.2ul instead of
the 2ul suggested in the Beckman protocol.
(B) TEMPLATE PREPARATION (Please refer to “Template Preparation” section of Kit Protocol)
1-DNA Template Purification:
-Qiagen kits are recommended by Beckman for this purpose (QIAwell and QIAprep isolation protocols for dsDNA and ssDNA, and
QIAquick gel-extraction protocol for PCR products). Other commercial kits like Promega Wizard can also be used. Standard alkaline
lysis preps must be used with no phenol. CsCl preps have been found to cause low signal and current instability problems.
-Beckman strongly recommends the use of agarose gels to check the quality of DNA before proceeding with the reactions.
-PCR products must be homogeneous. If you are having trouble getting good results even from an ultra-clear single band, try
purifying it with a GENECLEAN kit (from Alexis Corp).
-There must be no RNA in the DNA preparation.
-Remember that the new CEQ capillary system doesn’t tolerate the presence of salts nearly as well as the old slab gels.
-Template should be suspended in water or 10mM Tris-HCl pH 8.5, but NOT in either EDTA or TE (Tris-EDTA).
2-DNA Template Amount:
-As per the Note in the Kit protocol: “Determine the quality and quantity of template DNA by agarose gel electrophoresis.”
Quantitation should be conducted with a known standard.
-Be aware that accurate quantitation depends on careful purification. When the concentration is being measured on a
spectrophotometer the ratio of the signals at 260nm and 280nm must be 1.8 at least.
-Use the table in the Kit protocol to determine how much DNA you need. Be sure to make the conversion from grams to moles
based on the size of the whole vector, not just the insert.
-If you are screening a library be sure to factor in the estimated number of plasmids when calculating the molarity.
-The safest target amount of DNA is 100fmol. If you use 5pmol (see above) of primer this will give you a primer-to-template molar
ratio of 50:1 which is even better than the 40:1 suggested in the Kit protocol.
-Too little template (10 to 50 fmol) results in insufficient signal and difficulty normalizing peak heights after the dye front. Sequences
will have a lot of N’s.
-Too much (200 to 1000 fmol) results in overloading of the capillary and a delay in the start of the separation, making it hard for the
computer to establish a base line and analyze the raw data. Other consequences of too much template are irregular current traces, early
termination of reads, and potentially inaccurate base calling.
-If the target amount proves to be too little, try a series of samples 2X, 5X and 10X the original amount of template used. If the
target proves to be too much, try a series 1/2, 1/5, and 1/10 the original amount.
3-Template Pre-Heat Treatment:
-Pre-heat the template for 1 min @ 96*C.
-Please try this if your DNA is a plasmid and there is ANY chance that it is supercoiled. The pre-heating will denature the plasmid,
straightening it and releasing some of the impurities that adhere to it.
-This step must be done before adding the sequencing reagents.
-The initial step of the thermal cycling program below (20 sec @96*C) does NOT substitute for this pre-treatment.
(C) CYCLE SEQUENCING REACTIONS
1-“Preparation of the DNA Sequencing Reactions”:
-Please refer to the similarly titled section of the Kit protocol.
-Mix the ingredients for each reaction in 0.2ml thin-wall tubes, as per the Kit protocol, except that you should use enough primer
solution to provide at least 5pmol (3.2ul in the case of the 1.6uMolar M13 –47 primer that comes with the Kit).
-Run a pUC18 control template at the same time as your samples. Especially if you are a first-time user or have been having trouble
getting good sequence, this will help you tell the difference between sample problems and cycle-sequencing problems.
2-Thermal Cycling Program:
-The PCR machine should have a heated cover. The alternative use of an oil overlay can give rise to sample loss upon removal.
-The standard thermal cycling program in the Kit protocol takes about 2½ hours:
20 sec @ 96*C (denaturing temp)
20 sec @ 50*C (annealing temp)
4 min @ 60*C (elongation/extension temp)
for 30 cycles, followed by holding @ 4*C
-These standard conditions have been shown (by Beckman) to work well for MJ Research Models PTC 100 and 200, and for Perkin
Elmer Models 2400, 9600 and 9700. The use of other models may require the modification of conditions.
-Please be aware that the PCR machine needs to be programmed to suit the primer being used. If you are using a primer that has a Tm
estimated to be say 50*C then try an annealing temp of 45*C. A modified thermal cycling program that has worked for Brandeis folks
using T7 or other low Tm primers:
20 sec @ 96*C
30 sec @ 45*C
4 min @ 60*C
for 30 cycles, followed by holding @ 4*C
-If you are having trouble getting good sequences because there isn’t enough PCR product, one of the things you can try is
lengthening the program from 30 cycles to 40 or 50 cycles.
(D) POST-REACTION CLEAN UP
-The purpose of this step is to remove any leftover template, primer and DTCS reagent from the reaction product that is to be
analyzed. There are two ways to do this:
1-Ethanol Precipitation:
-This is the best method for retaining as much product as possible, so it is the best choice if your template concentration was on the
low side.
-Follow the directions in the Kit protocol, with these additional tips:
-In step (a), use 1.5ml microfuge tubes instead of 0.5ml. They are way easier to handle!
-In step (b), for multiple samples, instead of mixing only two components of the Stop Solution together and pipetting just 1ul of
glycogen over and over again – which is hard to do reproducibly because the glycogen solution is so viscous – try mixing all three
components of the Stop Solution together before adding to the microfuge tubes. This should provide more consistent results.
-In steps (d) and (e) use ethanol/water solutions that have been stored in a –20*C freezer and do the centrifuging at 4*C, but do not put
the reaction tubes themselves into the –20*C freezer because this brings down too much salt and that results in less signal during the
CEQ analysis. After each rinse-and-centrifuge step the supernatant liquid should be removed immediately.
-In step (f) vacuum dry with no heat.
-In step (g) be sure to use an aliquot of good quality formamide (aka Sample Loading Solution provided with the Kit) that has not
been cycled back and forth from refrigerator or freezer to RT, or otherwise allowed to oxidize or absorb atmospheric moisture.
-Store the samples at –20*C until submitted to the core facility.
2-Spin-Column Separation:
-This is the best method for removing as much reagent as possible and minimizing the dye front in the CEQ chromatogram which
often obscures the reading of the early part of the sequence, so it is the best choice if that part of the sequence is of interest to you.
-Be aware that there is more sample loss with spin columns than with ethanol precipitation, so you should start with twice as much
template.
-Instructions for the use of spin columns are provided by the manufacturers. DyeEx spin columns from Qiagen seem to work better
than CentriSep columns from Princeton Separations, and CentriSep columns work better than columns from Amersham.
-Beckman recommends pre-rinsing with de-ionized water.
-The final steps of this method are the same as with ethanol precipitation: drying, re-suspension in formamide, and storage at –20*C.
(E) SUBMIT SAMPLES TO CORE FACILITY FOR ANALYSIS
(F) MORE HELP CAN BE OBTAINED AT
(1) http://www.beckmancoulter.com/resourcecenter/literature/BioLit/BioLitList.asp?ProductCategoryID=DNAS
Once there, select “Technical Manuals” from the drop-down list. Three of them are of particular interest:
#390218aa “CEQ 8000 DNA Sequencing Customer Training Guide”
Chapter 5 on DTCS (Dye Terminator Cycle Sequencing) Chemistry
-Identical to Chapter 5 of #606948aa
-Has additional suggestions about pre-heat treatments, PCR machines and program
modifications, post-reaction clean-up techniques, etc.
#718119ab “CEQ DTCS Chemistry Guide”
-Has details about the reagent Kit and other required chemicals, additional
suggestions about primer design and template quantitation, etc.
#390216aa “Sequencing Analysis Troubleshooting Guide”
-Identical to #606947aa
-Suggests appropriate corrective actions for particular problems indicated by the
characteristics of the CEQ results.
(2) http://www.promega.com/pnotes/65/6921_27/default.html
This is an article entitled “Betaine and DMSO: Enhancing Agents for PCR” and describes the use of these additives to
improve the amplification of templates with high GC content and templates that can form secondary structure.
(3) http://www.beckmancoulter.com/Literature/BioResearch/NL-9150A.pdf
This is an article entitled “Higher Throughput at a Lower Cost” and describes the use of smaller amounts of DTCS
reagents: dilutions 1/2, 1/4, and 1/8 the amounts suggested in the Quick Start Kit protocol.