Designing an experiement to identify mutations in particular genes - (Feb/14/2013 )

Your first link explains everything about the master mix. You can optimize your reaction according to that. About the volume of the master mix, it depends on the total number of reactions. Mimumum volume for 1 PCR reaction in our lab is standard 50ul. I don't know if you can use the same. It all depends on lab to lab and how well some thing work in some labs. So you need to optimize the conditions that work best in your hands.

I have done similar experiments before, so from my experience I can say that:

<*>You can start with a 20ul reaction.
<*>Ideally set up a gradient to know which reaction is close to your desired PCR product
<*>You can then spend on time on optimizing your PCR product.

If you are planning to send out your PCR product for sequencing, you need to get (what I call) a crisp band (no visible smears/ unspecific bands). This increases your chances of getting a clean result.

Are you starting from scratch? or has someone in your lab done something like this before? If you are ordering primers yourself, you can try and get a uniform annealing temperatures across your target region. This way most of your reactions will need little/ no standardisation attempts.

Hope this is useful. Feel free to ask more questions. There are lots of people here who will help

I have a question about those cloning vectors, why do you need them?

In case of inherited diseases, there is possibility of only two alleles, so seqencing of PCR product is sensitive enough to catch heterozygotes. Usual approach is to look up all exons and amplify them and sequence separately, including exon-intron borders (so at least 50 bases on each end) that would suggest splicing mutations. Then you just sequence them. The requirement for sequencing varies, but usualy purified quantified specific PCR product is required. You need to find out the details of your sequencing service first, for example so you know what will the reading lengths be. Exons are usually around 400-500 bp long, but that depends a lot. Some sequencers can have reads only around 450 bp others up to 900 bp. You should know. You either need to design PCR products so small, or additional sequencing primers to sequence longer templates.

If you sequence acquired mutation for example in cancer, it's much more difficult, because they are in a minority. Usually highly sensitive detection approach is used, or depending on the percentage of tumorigenous tissue in the sample some other screening methods can be used. Sequencing is usually only reliable if the mutation burden in above 10 %. In that case you can use cloning and sequencing dozens of clones to catch at least one mutated clone, in that case you sent whole plasmids for sequencing (or depending on requirements) but in much higher concentrations than you would PCR amplicon.