Reason	Solution
Cells were stored incorrectly	•	Obtain new stock and store in liquid nitrogen. Keep the cells in liquid nitrogen until thawing
Home made freezer stock is not viable	•	Freeze cells at a density recommended by the supplier
	•	Use low-passage cells to make your own freezer stocks
	•	Follow procedures for freezing cells exactly as recommended by the supplier. Note the freezing procedure recommended by this handbook is a general procedure provided as a guideline only.
	•	Obtain new stock
Cells were thawed incorrectly	•	Follow procedures for thawing cells exactly as recommended by the supplier. Note the thawing procedure recommended by this handbook is a general procedure provided as a guideline only.
	•	Make sure that you thaw the frozen cells quickly, but dilute them slowly using pre-warmed growth medium before plating.
Thawing medium is not correct	•	Use the medium recommended but the supplier. Make sure the medium is pre-warmed
Cells are too dilute	•	Plate thawed cells at high density as recommended by the supplier to optimize recovery.
Cells not handled gently	•	Freezing and thawing procedures are stressful to most cells. Do notvortex, bang the flasks to dislodge the cells (except when culturing insect cells), or centrifuge the cells at high speeds.
Glycerol used in the freezing medium was stored in light (if applicable)	•	If stored in light, glycerol is gets converted to acrolein, which toxic to cells. Obtain new stock.

Reason	Solution
Growth medium is not correct	•	Use pre-warmed growth medium as recommended by the supplier.
Serum in the growth medium is of poor quality	•	Use serum from a different lot.
Cells have been passaged too many times	•	Use healthy, low passage-number cells.
Cells were allowed to grow beyond confluency	•	Passage mammalian cells when they are in the log-phase before they reach confluence.
Culture is contaminated with mycoplasma	•	Discard cells, media, and reagents. Obtain new stock of cells, and use them with fresh media and reagents.

Causes:

1. Too much template DNA
2. Excessive dilution of the BigDye reagent
3. Too little DNA
4. Too much primer
5. "Dirty" template DNA has been used.
6. Unsequencable region reached (eg. homopolymer G)


Solutions:

Check the concentration of the template DNA by gel electrophoresis. Do not rely on spectrophotometer readings alone as spec reading are often inaccurate, particular with plasmid templates.

Reduce the reaction scale rather than using a higher bigdye dilution. It is better to reduce the reaction volume rather than use very high dilution factors of the BigDye chemistry. For example, a 1:16 dilution of the BigDye (0.5 µl total) can be achieve with modest dilution (1:4) by performing the reaction in a total volume of 5µl.

Check to see if an unsequencable region has been reached. If an unsequencable or “hard stop” region is the cause then it may be possible to sequence though it by PCR amplifying the region using 7-deaza-deoxy guanosine triphosphate (7-deaza-dGTP), then sequencing the PCR product directly. The 7-deaza-dGTP analog disrupts the hoogsteen base pairing between successive guanosine bases and allow the sequencing DNA polymerase through the high G+C region. Check that the oligonucleotide primer concentration is correct. Do not rely on old primer stocks, especially those made up by your lab colleagues! Check that the template is clean. Consider sequencing a PCR amplified insert or switching to using a commercial plasmid miniprep kit if you are finding that many of your sequencing reactions are failing.

Causes:

1. Two or more templates were present in the reaction. This is the most common cause of mixed template.
2. A "double pick" of two colonies. This can when the colonies are too close together on the colony plate.
3. Two primers were present in the sequencing reactions. This can occur when using premixed PCR regents for sequencing where the primer stock is actually a mix of universal forward and reverse oligonucleotide primers.
4. The PCR fragment was not purified of leftover primes before sequencing.
5. Two priming sites are present in DNA template. This can occur when a PCR product with universal priming site tails is cloned into a plasmid.
6. Poor quality PCR template containing multiple DNA fragments was used.
7. Too low a primer annealing temperature was used in the sequencing reaction.
8. Different sequencing reactions were accidentally mixed at the clean up stage. This can also sometime occur if the same tip is used without rinsing.

Solutions:

Makes sure only one DNA template is present. Prepare a new plasmid prep making sure that only one colony is selected. If sequencing a PCR product check that only one PCR product is present by running an agarose gel. Remember that that even a relatively low amount of a small PCR product can cause mixed template problems. Check the template for possible multiple priming sites. If two site are present use a different primer. This can often occur when a fragment contain the priming is sub-cloned into a vector that also contains the priming site. Be very careful of this problem when using the M13 universal primers.

Insure that you use a PCR clean-up protocol that remove leftover PCR primers. Even low levels of the PCR primers can cause mixed signal problems, especially if they have a high annealing temperature.

Check the predicted melting temperature of the sequencing primer. If it is more than 5˚C above the annealing temperature used in the sequencing reaction then raise the annealing temperature. Because of the inclusion dITP in the BigDye sequencing mix the annealing/extension temperature can't be raised above 60˚C. If your primer is still misanneals at 60˚C then synthesize a new primer (this is easily done by removing bases from the 5' end until the Tm is below 60˚C).

Causes:

1. Poor quality DNA. Very common when sequencing plasmid miniprep templates.
2. Loss of the reaction during clean up. This can be a particular problem when using ethanol precipitation clean up protocols.
3. Too much template DNA. Excess template can kill the sequencing reaction.
4. Wrong primer used. More common than you might think!
5. Bad water. The water used contains a sequencing inhibitor.
6. Degraded or failed synthesis primer. Oligonucleotide synthesis is chemically complex and primer synthesis failures is fairly common.
7. Dead sequencing chemistry. Can occur if the BigDye chemistry is stored under the wrong conditions or is freeze-thawed too many times. Either the TaqDNA polymerase or dye labeled nucleotides can have degraded.
8. Blocked capillary. Every trace using a particular capillary fails. Can be identified by tracking trace quality on a trace by trace basis.

Solutions:

Poor quality DNA. The best way of avoiding this problem is to not sequence plasmid DNA and sequence a PCR amplified fragment of the plasmid insert. If this is not possible then it is recommended that a plasmid miniprep kit is used. One tip is to perform a final ethanol precipitation on the kit purified plasmid DNA. This often solves problems with the quality of the template.

Loss of sequencing reaction during clean up. This can be avoided by not using an ethanol precipitation protocol to clean up the sequencing reaction. There are a number of kits that work very well, unfortunately they can be very expensive. One tip for avoiding loss of the reaction DNA pellet when using the ethanol protocol is to add 1µl of a 20 mg/ml solution of glycogen (Sigma G-1508) to the sequencing reaction before adding the ethanol. This helps make the pellet visible and the glycogen does not seem to interfere with the injection of the sequencing fragments onto the sequencers capillaries.

Too much template DNA was used. This can be avoid by checking the concentration of the template on an agarose gel before sequencing. This will also allow you to see the purity of the template DNA and if there is a significant amount of contaminating genomic DNA or RNA present. Do not rely on a spectrophotometer reading to calculate the template concentration.

Wrong primer. This is simple to solve, but can be difficult to detect. Check the sequence of the primer and template to make sure that the primer binding site is present. This can be a particular problem with some "universal" forward and reverse primer sequences which do not work with some common plasmids. Do not trust other people's working stock solutions (especially those of your unreliable your lab colleagues!) and make your own. It might take 5 minutes longer, but it will save you a lot of future headaches.

Bad water. Inhibitors can end up in lab water stocks that can kill DNA sequencing reactions. If you think this may be a problem then throw out the water and use a fresh stock - remember water is cheap.

Degraded primer. Don't use old diluted primer stocks. Store the primers in 10mM Tris/ 0.1 mM EDTA (pH 8.5) rather than water. Don't use other peoples stocks. If you have any doubt about how the primer quality through it out and make up a fresh working solution from the primer stock.

Failed oligonucleotide synthesis. If you suspect that the primer is poor quality either have it presbyters or check in a polymerase chain reaction (PCR). Alternatively if you have a control template that you know should work with the primer then this can be a good way of identifying primer problems.

Dead sequencing chemistry. This is a relatively rare problem, however, if a batch of BigDye chemistry has not been used for some time, or there is any doubt about how it has been stored, then it is advisable to perform a control sequencing reaction before undertaking a large number of experimental reactions. Many problems with dead chemistry can be prevented by storing the BigDye chemistry in small aliquots and avoiding repeated freeze/thaw cycles.

Blocked capillary. Can be identified by tracking trace quality on a trace by trace basis. While this can be done manually we recommend using our QualTrace software. If you suspect that a sequencer capillary is blocked then inform the operator.

Causes:

Capillary overload. Unlike a lot of DNA sequencing problems delayed peak signal has only one cause - capillary overload. The two most common causes of capillary overload are too much template DNA or dirty template DNA contaminated with proteins an/or salt.

Solutions:

Use less template DNA. Modern ABI capillary sequencers require far less template DNA than the old slab based sequencers, however, many researchers are still using protocols developed for the slab based system. Try the suggestions made in the DNA sequencing protocol tips page.

Use cleaner DNA. Try changing you template preparation protocol. A good cheap template preparation protocol that works well with sequencing is the ExoI/SAP clean up.