Jerome:

you are exactly correct. For example, if your forward gene-specific primer was TGACTGACTGAC, you would use primer (GTTTTCCCAGTCACGAC)TGACTGACTGAC for amplification, where the M13F sequence is in parentheses, and after removing unused primer with exo or some other means, you would anneal the M13F primer GTTTTCCCAGTCACGAC for sequencing. In this way you could use the same sequencing primer with many different amplification primers and get more consistent sequencing results than if you use your gene-specific primer as both amplification and sequencing primer..

Not sure what you mean by "usual primer", but some people achieve more consistent sequencing when doing sanger sequencing of PCR products if you use the well-tested M13 primer, as opposed to the variation you often experience if you try a number of locus-specific primers as both PCR and sequencing primer. Having said that, I would recommend that the sequencing primer is just the M13F sequence, not the full primer used to amplify the locus (i.e., don't use M13F tailed primers for PCR and sequencing--just use it for the amplification and use a separate M13F primer for sequencing after removing unused amplification primer).

You can use the tail as matrix for sequencing. One example: you have PCR primers with many Rs, Ys, or Ks. By successful amplification, you have no idea about the correct sequencing primer. Using tailed primers, you can use the M13-Tail as matrix for sequencing, ignoring the original primersequence.

So, could I use M13f tailed primers for PCR, and then use a primer of only a M13-F tail for sequencing, no matter with which primer I've performed my PCR?

The M13tail (MMMM) is connected with the classical PCR primer (PPPP), so your complete PCR-primersequence is MMMMPPPP. However, there is one side-effect: when your PCR primer (PPPP) includes many wobbles, you have no fixed anealing temperature. Instead of this, you face a somewhat broader range, e.g. from 50-60°C. Anyway, this is easy to handle, just choose the the mid-temperature (here 55°C) for a first PCR. For decapods it works quite good. Otherwise you have to test different temperatures. The MMMM-tail is simply co-amplified and will be used as sequencing matrix.

Jerome:

you are exactly correct. For example, if your forward gene-specific primer was TGACTGACTGAC, you would use primer (GTTTTCCCAGTCACGAC)TGACTGACTGAC for amplification, where the M13F sequence is in parentheses, and after removing unused primer with exo or some other means, you would anneal the M13F primer GTTTTCCCAGTCACGAC for sequencing. In this way you could use the same sequencing primer with many different amplification primers and get more consistent sequencing results than if you use your gene-specific primer as both amplification and sequencing primer..

I'm currently attempting to use a pair of m13 tailed primers purchased from thermo Hs00836745_CE (google this) to sequence a BCOR mutation with amplicon of 252bp using ~200ng of gDNA, but am having trouble getting amplification of the specific region.  Due to the addition of the m13 tail the primers have high Tm (~78°C) and I'm using 2-cycle PCR due to this (No annealing step since the Tm is above the elongation temp).  I'm using Platinum Pfx dna polymerase which has a suggested elongation temp of 68°C.  So my thermocycler sequence looks like 1.) 94°C 4:45 2.) 94°C 00:15 3.) 68°C 1:00 4.) Go to 2, repeat 35x. 5.) 68°C 5:00 6.) 4°C Hold.  Anybody see any issues with what I'm doing, and have suggestions for optimization to help get the band?

@Eric,

I think you have to calculate the Tm for the initial PCR on the primer part that can actually hybridise to your template, eg only the gene-specific part. The M13 seq is NOT in your ene so won't contribute to annealing in the first couple of rounds.

@ all

remember to put different M13F and M13R on either end of your PCR product, not the same on both primary PCR primers. Otherwise you get mixed (R and F) sequencing mixed up.

@Henk-jan

If I want to use the M13 F and R in direct sequencing of m13 tagged product, I need to use (order) each primer in its reverse-compliment orientation.

How about use NGS for sequencing? If I plan to sequence mutiple-PCR product using NGS like Illumia MiSeq, is it necessary to apply M13 tailed primers for PCR? or just use mixed usual primers?

@ Nian-Hong

In Illumina sequencing (and other NGS platforms), the adapters already contain universal sequencing primers therefore M13 sequencing primer tails are not needed for sequencing PCR products. As others have stated in this thread, M13 tailed PCR primers have been used to facilitate the sequencing of many different PCR products (i.e., those generated with different primers) in the same run using Sanger sequencing.

@all

For anyone thinking about using M13 primer tails to sequence PCR products with a Sanger platform, there are a couple additional things to consider. First, many Sanger sequencing providers (e.g., Macrogen) have in the past supplied the M13 sequencing primers to sequence your M13 tailed products **free of charge**. This can help to lower your sequencing costs.

Secondly, note that there are multiple different possible M13 forward and M13 reverse sequencing primers that you can use to tail your PCR products. Although all of them have been proven to sequence well, it is essential that you exactly match the correct M13 sequencing primer with the one used as a tail on your PCR product. For example, if you used an M13 reverse (-27) primer as a tail, but then you use an M13 reverse (-29) primer as the sequencing primer, you will very likely create a bad mismatch at the primer-template junction, which will result in sequencing failures (i.e., useless chromatomgrams). While your PCR products should be tailed with both M13 forward and M13 reverse primers -- to allow you to sequence each product in both directions, you must be careful to use the same sequencing primer as your tail primer. I am attaching a figure from my book (Phylogenomic Data Acquisition: Principles and Practice) that illustrates this issue. In my book, I discuss in detail the use of M13 primers to sequence PCR products (Illumina sequencing is also covered). Sorry for the bit of self-promotion but I am not aware of any other review of this topic.

@Bryan and @all

Many thanks! I learn a lot from you guys!!

New paper about M13-tailed primers and the formation of primer dimers may be of interest to the people in this thread.

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