Problems with designing an experiment in identifying mutations of particular gen - (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!

Useful book: Molecular cloning: a laboratory manual, Sambrook et al.

bob1 on Thu Feb 14 19:47:22 2013 said:

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.accessexc..._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.mutationd...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!

Have a search for Roche Lab FAQs, they will provide a lot of the information you need.

bob1 on Sat Feb 16 06:53:09 2013 said:

Thanks for your recommendation!
May I ask one more question, if i am want to check whether a mutation of a gene do occurs in my sample by sequencing.
Is that I just need to carry a few steps, 1st extract dna and purifiy it, 2nd PCR amplificiation and purification and the last step is dna sequencing can already give me the results, without using the plasmid? since we can just analyze the code in the sequence and compare the code with a positive control. Thanks.

In theory yes, you can do sequencing directly off the DNA that you extract from the organism - however, if you further want to study the mutation (e.g. look at how it expresses in cells and what the effects are) then you need to clone it so that you have a permanent copy.

Doing PCR before the sequencing is not such a good idea as it could introduce mutations of its own. For example Taq DNA polymerase has an error rate of 10^-3, that is on average one base in 1000 will be incorrectly inserted in any PCR. If you think about how PCR works, this could be a problem if you then want to sequence.