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Sequencing TroubleshootingThere are a number of factors that can adversely affect the quality and length of sequence you receive. Geneservice have compiled a list of common problems we find after completing a sequencing job and where appropriate the services that we can offer to increase the chances of obtaining a satisfactory sequence from your precious template. You may also be interested in help on preparing sequencing templates. Secondary Structure Service DNA templates that have high GC content are particularly prone to the formation of secondary structures. For example, if a hair-pin loop structure exists in a template it can prevent the progression of the DNA polymerase along the growing strand and the resulting sequencing read can stop abruptly. It is widely known that conventional sequencing chemistry is unable to progress past some of these structures. Geneservice have developed an in-house reagent to combat the problem (more details).  If you would like us to try the Secondary Structure Solution (SSS) with your difficult templates please check the SSS box indicated on your online Sequencing Request Form. Template Rescue Service The quality of the template DNA you use in automated sequencing is of the highest importance. Templates that are too low in concentration or that contain sequencing reaction inhibitors such as salt, carbohydrate and or protein will all generate sequences low in signal intensity. When signal intensity is low the analysis software has difficulty in resolving the base peaks from background noise. Typically this results in a shorter read length with other random peaks being generated under the main data set. Geneservice recommends that plasmid templates are submitted at 100ng/ul and eluted in pure water. Geneservice offer a Template Rescue Service to enhance the quality and concentration of plasmid templates that have previously proven to be unsuitable for sequencing. The sequencing reads below shows analysed data with a low signal intensity and after the same template was rescued and resequenced using Geneservice Template Rescue Service.  If you would like us to try the Template Rescue Service (TRS) on your templates please indicate this on the online Sequencing Request Form. n-1 Primer Oligonucleotide primers can degrade to n-1 after multiple freeze thawing or this can be due to poor purification at manufacture. An n-1 primer will generate a second sequence lagging one base to the left or right of the main peak. Below is an example of a sequence generated with a mildly degraded n-1 primer. The main data is still visible but the detection of sequence variants in this example would be difficult. In more extreme conditions the data might prove completely unusable. If your custom primer is n-1, why not consider using one of our stock primers? These primers are successfully used every day in a large number of reactions and are thus proven to be free of n-1 variants.  Mixed Template In certain circumstances sequencing will fail because two or more populations of sequencing products are made in the same reaction and are impossible to properly resolve. This may be caused by there being mixed plasmids, or two or more template inserts present in the cloning vector, or there being more than one primer binding site. Multiple reads appear superimposed over each other. To stop this happening plasmid DNA extractions (or cultures for extraction) should always be prepared from a single bacterial colony. Ensure that PCR products have been cleaned before receipt so they are free from PCR primers which could also initiate extension in a sequencing reaction.  Sequencing a mixed population mixed plasmid clones will generate messy data from the insert site, prior to this (in the vector) the read might look perfect.  No Data NNNN indicates that the sequencing reaction has failed to make any extentsion products that are detectable during the electrophoresis.  This problem can be caused by the primer not annealing to the template or the template being of extremely low concentration or extremely low quality. Primer should be designed using the following guidelines: 18-22bp long, GC content of 50–55%, Tm of 55 to 60 degrees C. Dye Blobs Unincorporated dye terminators (commonly called “Dye Blobs”) appear at positions 70 to 80bp and again at approximately 100bp. The chromatogram below shows unincorporated dye-terminators superimposed over and partially obscuring the real peaks.  Dye blobs are caused by an imbalance of primer:BigDye:template. We use proprietary clean-up plates to remove dye blobs but in extreme cases of imbalance they can still remain. Geneservice have optimized primer:BigDye concentrations for specified template concentrations (below) and so it is important that you work to send the correct template concentration if you find that Dye Blobs are a problem for you.
Note: We require 5μl of sample and 5μl of primer per reaction. If dye blobs are interfering with your read then please let us know. We will remove them for you free of charge in the first instance and are happy to discuss optimizing your template concentrations so that this is not a problem for you. |
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