PCR optimization - (Mar/25/2007 )
Currently, I'm trying to optimize the amplification of a particular gene but this is my first time doing so I'm not sure on how to start. I hope someone can really help me in starting the experiment such as what is the suitable concentration and amount of reagents to use. Followed by, what do I vary (concentration/amount?) in the reagents used if I don't get the results.
Hmmm maybe the moderator can move this thread to the molecular biology thread. There are more experts over there.
Anyway, I will try to give my 2 cents.
Optimisation can be divided into two parts. The first one is the temperature to optimise annealing temperature. Second one is the concentration of the reagents.
Play around with concentration of your template, MgCl2, dNTP and primers.
The concentration, I will post it tomorrow. Getting late now...
Thanks! I'll be waiting for your answer.
First, you have to decide which polymerase you'll be using. For this it's important to know how long your gene of interest is, as some enzymes aren't good at amplifying longer targets. Another thing you have to bear in mind is the purpose of the amplification, if you'll have to clone it, and sequence clones or so, you'll be better of using a proofreading enzyme (Pfu and it's variants for instance). Next, compare prices among different companies if several have enzymes that will suite you.
If you're doing PCR, titrate you're Mg-concentration in 0,5 mM steps the first time (between 1,5 mM and 4 mM should be a range that's big enough), and see where you get the best results. Do you have a gradient cycler? With this you could titrate Mg and determine optimal annealing temp at the same time (optimal annealing temp is Mg-dependent).
Sorry for the late reply.
Here it goes.
For MgCl2, try from 0.5mM to 5mM. But I personally think around 1-2mM is sufficient enough.
For primer, the recommended concentration is 0.6uM however, play around from 0.5-1.0uM.
As for DNA template, try this range, 10pg - 1ug. But for my case, around 150ng is good enough.
Be careful with your unspecific binding.
Hope this helps.
Assume that you're using the commercially available PCR buffer which comes together with your Taq polymerase, it is easy for use to adjust only the MgCl2 concentration. Usually, we will start doing the MgCl2 optimization by preparing 5 different tubes-containing same concentration of PCR buffer, dNTPs, primers, and Taq, range from 1.0 mM to 3.0 mM MgCl2. As you may not have the optimised annealing temperature for your primers, you need to carry out gradient PCR. For the duration of denaturation, annealing and extension steps, it is very much depend on the length of your starting materials and PCR products. The duration of 30sec (95C) , 30sec (?) and 1 minute (72C) for respective step is good enough for most of the PCR amplification.