CEQ-A-0009
A PPLICATION I NFORMATION
Genetic Analysis: CEQ™Series
Long Sequencing Reads on the CEQ
CEQ 2000XL, CEQ 8000 & CEQ 8800 DNA Sequencer
Jim ThornPhD
European Applications Support Team
Beckman Coulter Europe
Notes
The CEQ 2000XL and CEQ 8000 use a combination of a 33cm capillary length and linear
polyacrylamide (LPA) gel to run all applications. In the CEQ 8000 these range from SNP analysis by
primer extension to sequencing reads in excess of 750bp with standard method LFR-a. The WellRed
dye terminator sequencing kits from Beckman Coulter are designed to give sequence reads in the
region of 1000bp. In this Application Note a modification of the LFR methods is described that is
capable of reading to over 1000bp at an accuracy of greater than 98.5%.
This method was developed with the reaction products from the control pUC18 and M13 -47 primer
generated from the standard Quickstart kit. The read length achieved be taken as a guide for other
samples. The longest reads will be achieved with the correct quantity of high quality DNA and primer,
as described in the Quickstart and DTCS kit protocols.
The work described in this Note is for guidance only and has not been fully validated.
Protocol
1) The pUC18 and M13 -47 primer were cycled according to the standard kit protocol.
a) A PTC200 thermal cycler from MJ Research was used
2) After cycling, samples were cleaned by ethanol precipitation in the CEQ sample plate
a) The plates were spun in an Allegra 21R centrifuge with S2096 rotor
3) The pellet was vacuum dried or fifteen minutes and resuspended in 40ul of SLS
a) A Drop of mineral oil was put on top and the sample plate loaded in the CEQ
4) The samples were run on the method 3KV 3hours, as detailed overleaf
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Applications Team Europe
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Jim Thorn
A PPLICATION I NFORMATION
Genetic Analysis: CEQ™Series
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5) The following modifications were made to the LFR-a method:
a) Initial voltage was set to ramp to 1.5KV over five minutes
b) The voltage was run at 1.5KV for five minutes
i) This initial low voltage is designed to help longer fragments enter the gel
c) The voltage was then ramped to 3.0KV over five minutes
d) The separation was run at 3KV for 180minutes
Results
Accuracy of base calling as reported on the CEQ by alignment with pUCdG
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Applications Team Europe
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Jim Thorn