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Center for Protein and Nucleic Acid Research

DNA Sequencing Troubleshooting

Good quality preps are very important!

Qiagen mini-preps are strongly recommended.

Primer sequences should be 100% specific and have no dimer formation. Please check primers for secondary structure.

Primers used in PCR reactions are NOT recommended for subsequent sequencing.

Primers should have a Tm close to 60 degrees.

Controls are always run for comparison and quality control.

Fluorescent thermal cycle sequencing conditions are:

  • 96 degree denaturation
  • 50 degree anneal
  • 60 degree extension
  • 25 cycles

Blank Samples? Possible reasons and remedies for blank samples.

a) Sample contains inhibitors, such as too much salt, EDTA, certain dyes, heme compounds. Remedy: Apply sample to Amicon Microcon-100 columns (Note for smaller DNA fragments you may need other columns with a lower MW cutoff) and follow our DNA Clean-up procedure. Some sample columns are available for you to try out. They are located in our office on the shelf above the computer at MB101.

b) Sample DNA template is degraded. Remedy: Run Agarose gel and check yield by A260.

c) Inaccurate quantitation. Quantitate sample in dH2O only. EDTA and other compounds can throw off the quantitation.

d) Primer choice not optimal or primer is degraded. Have you used the primer before? Is it really old? Too many freeze-thaw cycles will degrade primers.

e) Tm of primer is too low. Your primer should have a calculated Tm around 58-62 C.

f) Lastly, excessive supercoiling may be the culprit. Try submitting the PCR product or linearizing it with restriction digestion.

Too Light samples.

If your sample was too light. This can be caused by too little DNA, primer, or poor primer binding, although it is usually caused by low quality DNA. Try desalting the sample as discussed above in the blank samples section.

Mixed Samples

This is usually caused by multiple priming sites or multiple primers in the sample. If a sample has a mixed spot, but the rest of the sample is clean, this means that there is a mixed colony or multiple templates. The mixture typically starts at the cloning site and consists of one clone with an insert and one without the insert.

Falling off Samples.

Data starts out strong but dies off. This is typically caused by samples that are either too salty or contain too much template. Try our DNA cleanup procedure and quantitate the sample in dH2O only.

Short

This could be a PCR product or there are strong secondary structures or hairpin loops that cause the enzyme to fall off abruptly and stop extending. This can sometimes be overcome by adding DMSO. Resubmit your sample and check "GC-rich".

Peak Shapes are Poor (too broad)

This will cause the basecalling software to fail. This can mean either the sample contains some contaminant (i.e., protein or phenol) that makes the bands migrate poorly OR may be a result of too much DNA template. Remedy: clean-up the samples or make sure quantitation is accurate.

Rough Spot

This means there was a rough spot in the sequence, such as a run of one nucleotide or a poly TA region, and the read following such region has problems. Sequencing poly TA regions may be problematic.