eLabNotebook > Genomic Analysis > DNA Sizing on the CEQ™

Highly Precise DNA Sizing on the CEQ™ 2000 Fragment Analysis System

Keith Roby, Mark Dobbs, Doni Clark, Scott Boyer, and Graham Threadgill
Beckman Coulter, Inc.

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Irreproducibility, Short Readlengths, Inaccurate base calling, Data not analyzed

RAW DATA SIGNAL / CURRENT PROFILE POTENTIAL SOURCE OF PROBLEM POSSIBLE SOLUTIONS
Low Raw Signal and Normal current profile
  • Not enough DNA
  • Poor Thermal Cycling
  • Problem with Thermal Cycler
  • Bad Formamide
  • Incorrect primer concentration
  • Incomplete resuspension of pellet in formamide
  • Problem in post reaction clean-up of samples
  • Incorrect mineral oil
  • No mineral added
  • Increase the [DNA], always quantify the DNA
  • Check Primer Concentration
  • Make sure that the pellet is properly resuspended in formamide

    OR
  • If the pUC18 Control Template does not meet the specifications:
  • Use supplied SLS for sample resuspension
  • Check Thermal Cycler
  • Check that the correct method was chosen on the thermal Cycler
  • Problem with Ethanol precipitation
  • Check that glycogen was added
  • Centrifugation at 4°C
  • pH of Sodium Acetate
  • Concentration of EDTA
  • Check Mineral Oil was added to samples in plate
Low Raw Signal and Erratic or Crashed current profiles
  • Pre-heat treatment on
    plasmid samples was
    not performed
    (plasmid/cosmids/
    YAC/BAC/PAC samples)
  • The pre-heat treatment was carried out with all the sequencing reagents added
  • The cycling program had an extra step in the beginning for the preheat treatment
  • Too much DNA loaded onto capillaries
  • Sample contains contaminating ions that are injected onto capillaries- (ions from contaminating salts, bad formamide etc.)
  • Incorrect method of template purification e.g.- CsCl, Phenol/Chloroform, or insufficient removal of Ethanol during template preparation etc.
  • Preheat all plasmids templates
  • Refer to the CEQ 2000 DTCS chemistry Protocol, 718119-AB, pg. 18 and Application Note A-1872A for detail on the pre-heat treatment procedure
  • Pre-heat ONLY the DNA and WATER
  • Adding an extra step in the beginning of the cycling is of no benefit
  • Reduce the amount of DNA - follow protocol recommendations
  • Check method of DNA purification
  • Use supplied SLS for sample resuspension

Irreproducibility, Short Readlengths, Inaccurate base calling, Data not analyzed

RAW DATA SIGNAL / CURRENT PROFILE POTENTIAL SOURCE OF PROBLEM POSSIBLE SOLUTIONS
No signal associated with normal Current Profile (No signal even from the Unincorporated dyes)
  • Lost Pellet
  • Tips of capillary broken
  • ddNTPs not added
  • Sample not resuspended in formamide
  • Not enough sample in well (though in most cases will see a current problem)
  • Use "gel loading pipette tips" to remove ethanol during precipitation
  • Replace capillary array if tips are broken
  • Check that all reagents were added
  • Only use SLS for sample resuspension
  • Use at least 30 µl of SLS to resuspend the samples
Only signal of the Unincorporated dyes observed with a normal Current Profile
  • Failed Enzymatic Reaction
  • Incorrect primer
  • Incorrect annealing temperature
  • Did not add one of the reagents in the chemistry set-up
  • Broken Thermal Cycler
  • Check that all reagents were added
  • Check Tm of primer and work out the correct annealing temperature
  • Check the Thermal Cycler program for correct program and ramping
  • Check Thermal Cycler
No signal associated with bad Current profile See above explanation on bad current profiles.
Identical Erratic or Crashed Current profile in:
  • All eight capillaries of the array









  • One capillary
  • Air bubbles coming from the Manifold end
  • Manifold area dirty thus not allowing for the manifold end of the array to be sealed correctly
  • The current can be affected in all eight capillaries if not enough buffer is added to the buffer plate

  • Air Bubbles in the Sample Well
  • A sudden spurious dip is most likely due to an air bubble in the sample. Dips associated with DNA templates do not tend to be as sharp a peak.
  • Do 2 manual Manifold Purges and 2 manual Gel Fills from the Direct Control section of the Run module and rerun the samples (Note: use a fresh buffer plate)
  • Clean Manifold area of instrument
  • Fill buffer plate well at least 3/4 full


  • Check sample plate for air bubble before loading onto CEQ 2000
Jump in the Raw Signal baseline
  • Some sort of carry over in the sample from
    • Salt
    • Dirty Primer
  • Elute DNA template in sterile water or 10mM Tris pH 8.5 solution
  • Do not use a strong buffer (ionic strength) for elution of DNA in template purification
  • Use HPLC purified primers
  • Use correct concentration of primers
  • Do not add salts of any kind to the sample

High raw signal but wrong bases called; Sequence contains inserted bases

RAW DATA SIGNAL / CURRENT PROFILE POTENTIAL SOURCE OF PROBLEM POSSIBLE SOLUTIONS
High Raw signal (off-scale) and normal current profile
  • Too many cycles
  • Very efficient cycling causing the raw signal to over-range the detector
  • Bad quality primer (n-1 primer)
  • Degraded primer
  • Reinject the sample for less time - 30 seconds instead of 60 seconds
  • Cut down the number of cycles
  • Improve quality of DNA synthesis

Effects of Formamide

RAW DATA SIGNAL / CURRENT PROFILE POTENTIAL SOURCE OF PROBLEM POSSIBLE SOLUTIONS
  • Loss of dyes
  • Overall reduction in signal intensity
  • No signal
  • And/or current problems
  • Formamide breaks down into formic acid and ammonia, and these ionic products compete significantly with the larger DNA ions for injection and thus cause reduction in signal intensity. In addition they can also cause degradation of the sample.
  • Use SLS for sample resuspension
  • Store and use the SLS solution correctly
  • The procedure for deionizing formamide (CEQ 2000 DTCS chemistry Protocol, 718119-AB, pg. 4-6) must be followed exactly

Primer Artifacts

RAW DATA SIGNAL / CURRENT PROFILE POTENTIAL SOURCE OF PROBLEM POSSIBLE SOLUTIONS
  • The analyzed data contains a lot of inserted bases
  • Peaks under peaks in the raw data and also in the analyzed data
  • More than 9 bases per min of raw data
  • Mis-priming
  • Mixed templates
  • Poor quality primer
  • Select annealing temperatures that limit the annealing of mismatched primers
  • Unambiguous sequence regions should be chosen for selecting the priming sites
  • Prepare the DNA template in such a manner to avoid mixed template contamination
  • Use a primer that binds to a site internal to the primers used for PCR*
  • In some instances the contaminating templates is present in very small quantities and dilution of the sample helps to decrease the concentration of the contaminant even further

Salt mobility

RAW DATA SIGNAL / CURRENT PROFILE POTENTIAL SOURCE OF PROBLEM POSSIBLE SOLUTIONS
Compression of bases in the region of 20 – 70 bases from the primer
  • Caused by the migration of buffer
  • Only see in software V1.1
  • Not present in V 2.X or greater

Poor resolution

RAW DATA SIGNAL / CURRENT PROFILE POTENTIAL SOURCE OF PROBLEM POSSIBLE SOLUTIONS
Check the accuracy of base calling with the pUC18 Control Template
  • Poor Cycling
  • Bad Formamide
  • Problem in post reaction clean-up of samples
  • Incorrect mineral oil
  • Capillaries
  • Gel
  • Check pUC18 Control template – if this is OK then the DNA template and/or primer is at fault

    Or
  • If the pUC18 Control Template does not meet the specifications:
  • Use SLS solution to resuspend samples
  • Check Thermal Cycler
  • Problem with Ethanol precipitation
  • Check that glycogen was added
  • Centrifugation at 4°C
  • pH of Sodium Acetate
  • Concentration of EDTA
  • Check Mineral Oil was added to samples in plate
  • Check the life of the capillaries
  • Check life of gel

Capillaries

RAW DATA SIGNAL / CURRENT PROFILE POTENTIAL SOURCE OF PROBLEM POSSIBLE SOLUTIONS
  • Resolution is bad
  • Less than 6 peaks per minute
  • Spurious peaks
  • Late start of data even though current is normal











  • One or more of the four dye traces are >6000 counts in the beginning of the run
  • The capillary has done more than 100 runs
  • The capillary coating has been damaged
  • The tips were not kept moist
  • The array was not stored correctly
  • Some type of chemical was used to load the sample that destroyed the coating
  • A sample plate was set up but no corresponding buffer plate was loaded


  • Dirt on the window
  • Manifold area dirty
  • Purchase a new array















 

  • Remove the capillary array at the manifold end and clean capillaries. Use sterile water or absolute ethanol. DO NOT use methanol. Clean in one direction only.
  • Clean Manifold area

* All trademarks are the property of their respective owners. Where applicable, the PCR process is covered by patents owned by Roche Molecular Systems, Inc., and F. Hoffman-LaRoche, Ltd.

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