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

I am designing an experiment to identity mutations in particular gene of a disease by using plasmid cloning vectors, sequencing techniques.
Firstly, I am going to extract the dna from myself and then amplify it through PCR reactions.
I would like to ask how can i determine what concentration and volume of each components in order to prepare a suitable PCR master mixture for the PCR reaction, (my PCR master mix should contain taq polymerase, dNTPs and MgCl2)
and how much PCR master mixture and my dna sample (what concentration is the best?) should be added in order to obtain a PCR product?
On the other hand, at the end, I need to give my sample out for DNA sequencing, should I just give the pure DNA sample or the recombinant plasmid for analysis?
Would you please recommend me some useful books or websites for designing this kind of experiment (especially on how to determine suitable concentration and volume for preparation of buffers, master mix etc).
Thank you for your help!

-helpwithdna-

student guide for PCR optimization-

http://www.babec.org/files/PCR_2012/PCR_Optimization_Student_Guide_2012.pdf

and PCR calculator-

http://www.sigmaaldrich.com/life-science/molecular-biology/pcr/learning-center/pcr-tools.html

-neuron-

neuron on Fri Feb 15 07:39:41 2013 said:


student guide for PCR optimization-

http://www.babec.org..._Guide_2012.pdf

and PCR calculator-

http://www.sigmaaldr.../pcr-tools.html


Thanks for your reply. I would like to ask, how can I determine the suitable concentration of each component in PCR master mix?

I found a web about PCR master mix (http://www.accessexcellence.org/LC/SS/PS/PCR/PCR_technology.php)
I still not quite sure about the calculations. Is all those calculation based on the concentration of stock solution provided by the lab?
If i have to decide the optimal concentration and volume for the PCR master mix, how can I do? Is there any criteria?
Can I just apply the result of this calculator for my master mix? http://www.mutationdiscovery.com/md/MD.com/screens/optimase/MasterMixCalculator.jsp?action=none
I am not sure about the optimal reaction volume for my PCR. In my previous experience, our PCR reaction(with template DNA, primer, master mix, water) is equal to 15ul, but I dont know the reason for choosing 15ul. Thanks so much for your help!

-helpwithdna-

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.

-neuron-

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 :)

-Ameya P-

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.

-Trof-