Using a good primer is essential to obtaining the best quality DNA sequencing read. There are many variables to consider when choosing a primer and the information presented below will hopefully help customers to achieve the best results possible.

Plasmid Considerations

When choosing a primer to use with a particular plasmid, it is very important that:

• A priming site exists within the plasmid...
• There are no mis-matches (esp at 3' end) between primer and binding site...
• There is only one priming site in the plasmid...
• The priming site is at least 30 bases away from the sequence of interest...

Often the reason for a complete failure of the sequencing reaction is because there is either no primer binding site, or there is a mis-match. Mis-matches at the 5' end can often be tolerated, since it the 3' end of the primer that is the "business" end. Multiple peaks of sequence at every position can indicate that more than one priming site is present (this could be a true second site, or a pseudo site that resembles the proper priming site. You should also be aware that the first 30 or so bases after the primer are not seen in the sequence output. This is because those sequencing products are not well recovered in the cleanup stage.

Establishing Plasmid / Primer Compatibility

So, how does one establish that a primer is compatible with a particular plasmid? If the plasmid is a standard cloning plasmid that is well characterised, then probably you will have a map of the plasmid to hand and that will contain the information you need. If you intend on using any of our standard primers, we would still recommend checking that the sequence is actually the same. We have a primer / plasmid compatibility page that might help.

If you do not have immediate access to detailed plasmid information, then the world wide web is your next port of call. Do a "Google" search with the name of the plasmid you are working with. Most commercial suppliers will have detailed information (often including the full sequence) for their plasmids on their web sites. They will also usually provide information on which standard primers work with their plasmids or provide the sequence of custom primers that work (they possibly sell these primers too). If you can't tell from the plasmid map which primer is right, download the sequence and put it into a DNA manipulation program. You can then align different primers to the sequence and see which ones align and where they are relative to your cloning site.

If your plasmid is not a commercially available one, you could try asking whoever supplied it to you. If you still can't get any information, you could try various standard primers in the hope that they will work (but this could get pricey...) or you can use an internal primer within the insert (assuming you know some of the sequence or what the sequence should be) to sequence out into the plasmid. Once you have this sequence, you can check to see if any standard priming sites are present or design your own primer.

Primer Design Considerations

This is obviously not a problem if you are using established sequencing primers, since they have been optimised already. However, if you are designing your own primers, good design is essential. To achieve good sequence, you should always follow these guidelines when designing sequencing primers:

• Always design your primers using known/good quality sequence
• Aim for a Tm of about 60 degrees celcius [Tm=(sum A+T) x 2 + (sum G+C) x 4]
• Try to have about 50-55% GC content
• Have at least one G or C at the 3' end of the primer (but not GGG, etc.)
• Avoid homopolymeric regions (e.g. CCCCC)
• Avoid repeats of sequences (e.g. GATCGATC)
• Avoid any self-complementary regions (e.g. GATCNNNGATC)

Anyone familiar with PCR primer design will recognise the above rules. Cycle sequencing is similar to PCR (except only one primer is used) and so often primers that work for PCR will work for cycle sequencing. However, primers that have excessively high Tms (over 65 Deg. C) may generate high backgrounds, when used for cycle sequencing. Primers with low Tms (below 55 Deg. C) often give weak sequence. This can also be seen with certain "standard" sequencing primers such as T3 and SP6. For this reason these primers have had to be altered for automated sequencing.

Primer Synthesis Considerations

Primers nowadays are cheap and of generally high quality. We seldom see issues associated with primer synthesis problems. However, it does happen. The most common is problem is associated with poor coupling efficiency in the synthesis reaction. This leads to truncated primers (N-1, N-2, N-3, etc) being present in the final prep. Since oligos are synthesised from the 3' end, these truncations are present at the 5' end of the oligo. When this mix of oligos is used for sequencing, what is producd are sets of products that all terminate at the same position by virtue of the di-deoxy terminator present, but which are N, N-1, N-2, etc long. The N-1 product co-migrates with the full length product produced by di-deoxy termination at the preceeding base in the sequence and so results in two peaks being present (assuming that the terminating bases are different). If you're having problems visualising this, write it down on a piece of paper. Also, you can go to our trouble-shooting section, where we have a beautiful example of this.