Frequent Reasons for Failure to Get Good Data
Failure results when there is an insufficient level of fluorescent termination products for the computer software to assign a sequence. Some possible reasons:
- Our new capillary sequencer (and corresponding new sequencing polymerase kits) provides longer reads but is also more sensitive to residual salt and ethanol in sample.
If you are experiencing variable results(e.g. in silica based purification schemes where 10 mM Tris is the recommended elution buffer), try a post spin-out ethanol PPT or elute in water instead of E.B.
- Too little template results in reactions with little or no signal and poor or no base calling.
- Too much DNA producing reactions which terminate prematurely, often with fewer than 250 bases of reliable sequence data.
- Poor quality template DNA. Template DNA must be free of residual ethanol and salt.
- Insufficient primer concentration or poor quality.
- The Tm of the primer is << 50°C.
- The template does not contain a sequence complementary to the primer.
- Primer and/or template was not added to the reaction.
- Using the same primers for sequencing as were used for PCR. One of the primers used to generate the PCR product does not work under fluorescent cycle sequencing reaction conditions.
- Template from two different colonies was submitted inadvertently. You may have picked the biggest colony or plated too densely in which case many of your colonies are actually two different colonies rather than the one of interest. This results in two overlapping sequences on the electropherogram. The fix is to re-plate at a lower density and re-sequence.
Some Common Mistakes with Qiagen Plasmid Kits which Affect the Sequence Quality
The directions for cell growth are not followed resulting in overloading the Qiagen resin. Usually, a poor yield of plasmid DNA results, presumably due to competition with RNA fragments for binding to the Qiagen resin. Use the recommended quantity of LB broth (don't use Terrificbroth) for cell growth.
The isopropanol-precipitated DNA is not washed with 70% ethanol to remove excess salt. Wash the DNA pellet at least once as directed with 70% ethanol. Residual salt in the final template will interfere with the activity of Taq polymerase resulting in sequence data which extends fewer than 200 bases from the primer and exhibits a low signal to noise ratio.
The template DNA is not dried completely before final resuspension in H2O. To remove residual ethanol, dry the DNA for 5 min. in a properly operating speedvac. If air-drying, make sure that the DNA is dry (no fluid in the tube, the DNA pellet does not look wet). When air drying, a brief 15 min incubation of the open tube at 65 oC is often sufficient to completely dry the DNA. Residual ethanol is detrimental to Taq cycle sequencing resulting in data with a drastically reduced signal.
DNA Sequencing Troubleshooting Table
|Poor Quality Template|
|Weak , noisy signal. Signal strength in raw channel is usually less than 100||Contaminated Template:
Template DNA must be free of residual ethanol and salts. And also free of cellular components such as RNA, proteins, polysaccharides, and chromosomal DNA
|Large stop peaks||Degraded DNA||Re-isolate DNA|
|Multiple, overlapping peaks||More than one template present||Clean up PCR products
|Double pick of two colonies||Re-isolate DNA|
|Large dye blob at ca. 60 and 100 bases||Insufficient template||Increase amount of template|
|Primer Related Problems|
|No sequence generated||Insufficient Template||Increase amount of template|
|Contaminant||Clean up template|
|Insufficient primer||Increase amount of primer|
|Primer has no annealing site
Poor primer design
|Noisy data with weak signal||Not enough DNA||Use more DNA in reactions|
|Primer anneals poorly||Re-design annealing temp|
|Noisy data with good signal strength||Contaminated template||Clean up template|
|Multiple templates||Examine template on agarose gel|
|Multiple priming sites||Redesign primer|
|Multiple primers with PCR products||Purify PCR template|
|Too much DNA||Use less DNA|
|Noise up to or after a certain point||Mixed plasmid prep/ Multiple PCR products||Ensure only one template present|
|Long homopolymer A or T (slippage)||
(includes data from ABI 3730 Sequencing Chemistry Guide “Appendix B: Troubleshooting”)