| Symptom | Possible Problem | Possible Cause | Potential Solution |
| Other than background, no raw data signal is observed in any of the four channels for the entire plate (Fig. 5.1). | Failed injection of the entire plate. | Operator Error. Buffer plate may have been used for injection instead of sample plate. | Take care to use proper plate and repeat injection. |
| | Bad template. | Template DNA may be non-existent or of extremely poor quality. | Re-prep the template DNA and repeat DNA sequencing, and consider another template preparation method. |
| | Integrity of reagents may be compromised. | Polymerase has no activity. | Check reagent integrity by running a reaction with control template DNA and primer. |
| Other than background, no raw data signal is observed in any of the four channels for some individual capillaries.
(Fig. 5.1). | Failed injection. | Failure of capillary and/or electrode to make contact with the sample during electrokinetic injection. | Check status of electrodes. If damaged call Field Support at 1-800-743-7782 (USA) or your local representative.
Check the cathode and anode ends of the capillary arrays. Replace broken arrays. |
| | No or very low capillary current. | Bad capillary. | Check number of runs on this set of capillaries. Arrays may need to be changed. |
| Generally low signal in sequencing data.
(Fig. 5.2 and Fig. 5.3) | Poor electrokinetic injection. | Electrokinetic injection was performed for too short a duration. | Try increasing the injection duration by 50 to 100%. |
| | Poor electrokinetic injection. | Sequencing reaction is contaminated with negatively charged species such as protein, or salt ions. | Cleaner template preparation method may be necessary; G-50 Sephadex post-reaction clean up may help |
| | Poor sequencing reaction | Insufficient amount of template DNA used | Increase amount of template DNA
(Fig. 2.2B) |
| | Poor sequencing reaction | Insufficient amount of primer was used. | Check primer concentration and increase the amount of primer used
(Fig. 2.4 for dye primer). |
| | Poor sequencing reaction | Poor annealing of the primer. | Annealing temperature used may be too high. Try lower annealing temperature. |
| | Poor sequencing reaction | Insufficient number of cycles used in cycle sequencing. | Increase cycles to maximum of 35. |
| | Poor sequencing reaction | Template preparation may contain a substance that inhibits polymerase. | Cleaner template preparation; ethanol precipitation or G-50 gel filtration prior to sequencing. |
| | Poor sequencing reaction | Template may contain too much EDTA | Resuspend template in water or buffer containing less than 0.1 mM EDTA. |
| Dye Primer Sequencing: Primer peak without sequencing data
(Fig. 5.11) | Primer failed to anneal | Annealing site for primer does not exist in template DNA. | Check the sequence of the annealing site and primer and repeat DNA sequencing. |
| | Primer failed to anneal | Cycle conditions were not suitable to facilitate annealing of primer. | Check thermal cycling program and sequence of primer to establish correct annealing conditions. |
| | Bad template | Template DNA may be non-existent or of extremely poor quality. | Re-prep the template DNA; consider another preparation method. |
| | Reactions performed incorrectly | Operator Error. One or more sequencing reaction components may have been left out of the reactions. | Take care to add each component according to accompanying protocol and repeat DNA sequencing. |
| | Polymerase activity. | Polymerase has no activity. | Check reagent integrity by running a reaction with control template DNA and primer. |
| Dye Primer Sequencing: Signal in one or more channels is weak or non-existent (Fig. 5.10) | Termination reaction pooling error. | Four reactions not pooled correctly (not done in dye terminator reactions). | Repeat DNA sequencing. |
| | Incorrect dye primer reaction set up. | One or more components may have been added incorrectly or left out | Take care to add each component according to accompanying protocol (Dye Primer Sequencing Protocol) |
| | Instrumentation error | Beam splitters and band pass filters may be installed incorrectly. | Consult MegaBACE Users Manual and call Field Support |
| Dye Terminator Sequencing: Terminator blobs without sequencing data (Fig. 5.16). | Bad template. | Template DNA may be non-existent or of extremely poor quality | Re-prep the template DNA; consider another preparation method. |
| | Primer failed to anneal. | Primer has no annealing site on the template DNA. | Check the sequence of the annealing site and primer and repeat DNA sequencing. |
| | Primer failed to anneal. | Operator error. Primer was not added to sequencing reaction mix. | Take care to add primer according to accompanying protocol and repeat DNA sequencing. |
| | Primer failed to anneal. | Cycle conditions were not suitable to facilitate annealing of primer. | Check thermal cycling program and sequence of primer to establish correct annealing conditions. Repeat sequencing. |
| Dye Terminator Sequencing: Terminator blobs in the sequencing data
(Fig. 5.17) | Insufficient removal of unincorporated dye terminator from sequencing reaction products. | Poor dye terminator clean up. Unincorporated dye terminators were co-precipitated with DNA sequencing products. | Review the dye terminator sequencing protocol and follow recommendations for ethanol precipitation (ET Dye Terminator Protocol, Thermo Sequenase II Dye Terminator Protocol). Perform ethanol precipitation without cold reagents and cold incubations as per protocol provided with dye terminator kit; use recommended EtOH concentration and included ammonium acetate |
| | Unincorporated dye terminator in the sequencing reaction products. | Some operators report that Thermo Max or "Microwave" prepared DNAs are more difficult to clean up than others. | Consider further clean up of template DNA. For example, prior to sequencing try treating template DNA with an ethanol precipitation or G50 column. Consider using a cleaner template preparation method such as alkaline lysis with or without column clean up. |
| Delayed start of raw sequencing data (Fig. 5.4 and Fig. 5.5). | Overloaded capillary. | Too much template DNA was injected into capillary causing a drop in capillary current and a subsequent loss of resolution. | Increase the resuspension volume of the DNA sequencing products by 100-200% and re-inject the sample using the original injection parameters (Fig. 5.7). |
| | Overloaded capillary. | Too much template DNA was injected into capillary causing a drop in capillary current and a subsequent loss of resolution. | Repeat injection of these samples using a shorter duration (25-50% shorter) or a lower voltage (25-50% lower). |
| | Overloaded capillary. | Too much template DNA was injected into capillary causing a drop in capillary current and a subsequent loss of resolution. | If injecting from water, try evaporating samples to dryness and dissolving in MegaBACE Loading Solution (70% formamide, 1 mM EDTA). |
| | Overloaded capillary. | Too much template injected into capillary causing a drop in capillary current. | Repeat sequencing using less template DNA. |
| | Low capillary current | Bad capillary. | Check number of runs on this set of capillaries. Arrays may need to be changed. |
| Peak spacing changes during run (‘accordion effect’)
(Fig. 5.8 and Fig. 5.9) | Current fluctuation during the run | Capillary nearly overloaded | Increase the resuspension volume of the DNA sequencing products by 100-200% and re-inject the sample using the original injection parameters |
| | Current fluctuation during the run | Bubbles may form in the LPA matrix either during sample injection or during electrophoresis. | Try re-injecting the sample again. |
| Strong, but poorly resolved peaks at the beginning of raw sequencing data | Overloading capillaries with sequencing products | Strong sequencing reaction | Using the same injection voltage, reduce the duration of injection by 25-50%.
Using the same injection duration, reduce the voltage by 25-50%. |
| | Overloading capillaries with sequencing products | Strong sequencing reaction | Increase the resuspension volume of the DNA sequencing products by 100-200% and re-inject the sample using the original injection parameters |
| | Overloading capillaries with sequencing products | Strong sequencing reaction | Repeat the sequencing reactions with less template DNA. |
