Preparation of samples
We need 5µl per reaction of both your primer and DNA at the concentrations specified above.
||1ng/µl per 100bp
||1ml of overnight culture
DNA concentration needed
- Plasmid: We require a concentration of 100ng/ul for plasmid samples.
- Purified PCR samples: The concentration of DNA required is dependent on the length of the sample sent. A concentration of 1ng/ul per 100 base pairs is required for successful sequencing.
We require your primers to be sent at 3.2pmol/ul. You can choose to send your own primers, use our stock primers or you can order custom primers that will be delivered to the sequencing laboratory. It is recommended when designing primers that you ensure you pick a suitable primer length of between 18 and 23 base pairs long, design your primers at least 50 bases upstream of your area of interest, a tm between 55°C and 60°C and ensure your primer has a GC content between 40 and 60%.
Quality of samples
All samples must be of a good quality, free from contaminants such as salts, other contaminating DNA, unincorporated dinucleotides (in PCR reactions) and other reagents that would interfere with the reaction.
It would be advisable to run your samples on agarose gels prior to submission to ensure that only one clearly defined band is visible on the gel and your DNA is not contaminated with other DNA and has not degraded.
It is important that you are aware of any motifs or secondary structures that might impede high quality sequencing results. If you know that this is most likely going to be a problem we recommend you use our secondary structure resolution option when ordering.
Most common reasons why sequencing reactions fail
Samples too concentrated
Figure 4 is the raw data from a PCR product that was too concentrated. As shown in the figure the results reach above the highest detectable levels on the graph. Figure 5 is the corresponding electropherogram for this sample. Note the peaks are poorly defined, overlap and are not uniformly spaced. The sample produced what would be known as a mixed trace, this would be due to the poorly resolved base calling in the electropherogram. Figure 6 shows a plasmid sample which is too concentrated, the sequencing trace is very concentrated at the beginning and it decreases rapidly producing a shorter than expected sequencing trace.
Low sequencing trace
Figure 7 and Figure 9 display failed sequencing traces. On the raw data (Figure 7) the scale is low and there is no obvious increase in fluorescence. Figure 8 shows the electropherogram for data from Figure 7 no bases are called and they are all assigned with the letter N. In Figure 9 the sample has failed due to a low concentration of DNA in the sample, there is some binding but at a low level of fluorescence. Figure 10 is the electropherogram for Figure 9, bases are not being called with a high quality of confidence.
Table 1: Potential causes for samples producing a poor sequencing trace
|Insufficient DNA concentration
||Increase DNA concentration
|Insufficient primer binding
|No primer binding site present
||Redesign primer or use different primer
||Re-extract DNA template or clean up template
||Make up new primer stock
Secondary structures are unexpected early terminations of your sequence (Figure 11). These can be due to your template having a high number of GC rich areas which have a tendency of causing the DNA to loop to form a hairpin bend. They polymerase cannot continue the reaction and therefore the sequence terminates early.
Table 2: Potential causes for early termination of a sequencing trace
|Secondary structure present in your DNA
||Choose secondary structure resolution option when ordering
||Redesign primers to avoid the formation of the secondary structure
Several different mixed traces can be produced dependent on why the sample has produced a mixed trace. If the trace has produced a mixed trace the whole way through the electropherogram (Figure 12) it is most likely due to either your sample containing more than one DNA template or your primer binding to more than one area of on your DNA.
Other mixed traces that could be produced could be due to mutations in your template. Single nucleotide polymorphisms (SNP) could cause what would look like a mixed trace at one particular base which does not affect the rest of the sequencing trace (Figure 13). An insertion or deletion in the DNA however will look like a normal sequence until the insertion or deletion occurs and then a mixed trace will occur as different sequences are now being produced. A mixed trace might occur due to enzyme slippage also, this can occur after a homopolymeric region (Figure 14), this enzyme slippage happens and the growing strand does not stay paired correctly with the template DNA during polymerization.
Table 3: Potential causes of mixed sequencing traces
|Mixed templates present in sample
|Frame shift mutation
||Use different primer after the mutation or sequence the complimentary strand
|Multiple primer binding sites
||Ensure the primer will only bind at one spot
|Enzyme slippage due to a homopolymeric region
||Sequence the complimentary strand
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. We have optimized primer:BigDye concentrations for specified template concentrations 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.