I am currently preparing a PCR mastermix for the first time, and have a few questions regarding the distribution. I am trying to amplify a specific gene cluster in a sample of DNA so I can use it in a cloning vector; however, I am not sure how many separate PCR reaction mixtures I should prepare in my mastermix for this purpose? Also, what is the purpose of a negative PCR reaction mixture (withouth the template DNA), as I saw a procedure recomend making one. Should I prepare the same number of negative reactions as positive, or should I prepare only 1?
A PCR Master Mix is just a way to speed up your pipetting. Instead of preparing 10 different reactions of 20ul each (for example) you prepare one reaction of 200ul and then you split it into 10 tubes. This also reduced distraction reduces errors (it is less likely to forget to pipet once 100ul that 20 times 5ul). Also, less plastic (pipet tips) is used, which for me is a good plus.
Given the error of some pipettes, it's generally good to count for an extra reaction every 10. For example, if you need to run 10 PCR, make a Master Mix for 11.
If you are planning to run the same PCR on different templates (which is often the case) then you don't need to add the template in your Mix.
Negative and positive controls should always be included.
The negative control of a PCR reaction is often done by substituting the template DNA with water. The negative control should not show any amplification band. This guarantees that you don't have DNA contaminants in your mix and that you primers do not amplify each other.
A positive control is a reaction that is expected to work. In this case, if your positive control shows amplification but your sample don't, you know it is not because of some component in the reaction behaving unexpectedly (Polymerase, buffer, etc..).
Let's make a practical example.
Let's say I want to amplify the same target sequence from 10 different DNA samples.
The 1X reaction looks like something like:
**PCR 1X** Template 1ul Primer F 1ul Primer R 1ul H2O 10ul MgCl2 2ul dNTPs 2ul Buffer 10X 2ul Polymerase 1ul
Now, considering the positive and negative controls, and 10% extra volume, I would prepare a Master Mix for 13 reactions. Of course, the template must be excluded.
**Master Mix 13X:** Primer F 13ul Primer R 13ul H2O 130ul MgCl2 26ul dNTPs 26ul Buffer 10X 26ul Polymerase 13ul
Now aliquot 19ul in the 12 tubes, add 1ul of the right template to each of them (in the C- just add 1ul H2O) and run it!
There are cases in which you may want amplify different targets sequences from the same DNA. In this scenario, you want to include the template in the Master Mix, since it is the same for all the reactions, but you want to add the primers separately since each reaction will contain a specific set of primer to amplify a specific sequence.
There are four things you can change depending on how much DNA you need to insert into the cloning vector:
- (i) amount of PCR reaction mixtures ("how many tubes")
- (ii) concentration of template DNA ("how much template DNA in a tube")
- (iii) how many cycles ("how many times will you amplify your template")
- (iv) how efficient amplification is ("how efficient is amplification") i.e. ideally each cycle exactly doubles your DNA, but in practice it's almost double or sometimes substantially lower than double
Remember, 1 tube after 10 cycles ideally contains exactly as much DNA as 2 tubes after 9 cycles. Usually changing the number of cycles is the way to go. You don't need that much DNA for vector insertion, I would go with a single tube to begin with.
How much DNA do I need?
We can't help you with determining how much you need, but see how much you need for inserting into the vector; make an excess using PCR, check to see whether the PCR worked on a gel (size purification) and measure the concentration of your product. Then you take the appropriate amount for making your vector.
Negative PCR control?
Negative control is a control reaction that contains all essential components of the amplification reaction except the template. This enables detection of contamination due to contaminated reagents or foreign DNA. Here you should see exactly no PCR product - if you do, you'll have to troubleshoot your reagents!
How many separate PCR reaction mixtures I should prepare?
If you're just amplifying DNA, one tube should be enough to see clear bands on a gel. In other cases, for instance with quantitative PCR (which you aren't doing), you'd prepare replicates, so you have a negative tube, and let's say 3 experimental tubes (technical replicates) so you can average across them for better accuracy and reliability.
When preparing a master mix, e.g. for 10 reactions, prepare it as if you were making 10% more reactions (11 reactions) so that you can comfortably pipette everything. You need 100mL of a solution for an experiment? Make 110mL. This is general good lab practice - always make a slight excess of master mixes.
The number of reactions you set up depends on a lot of parameters. To answer this question you'd need particulars about the source of template DNA, melting point of primers, the taq you are using and the size of the product you want to amplify. All of these will dictate how you optimize your PCR. Have a look at the below reference http://cshprotocols.cshlp.org/content/2009/4/pdb.ip66.abstract