A Cheatsheet for Optimizing your PCR

Starting off with optimizing a PCR can be a daunting task. Optimization outcomes vary widely with reaction temperatures, reagent consistency and sometimes even pipetting technique. The following guide will help you to optimize your PCR results in no time.

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1. Primer Selection

Primer selection is the first step towards a successful PCR is primer selection. There are two ways you can go with this. The first is to select primers that have been already reported in previous publications. It is a shortcut but care must be taken to select primers that have been reported in a large number of current publications. 

The second way that you can go around is with designing a primer of your own. Online programs such as the primer designing tool by NCBI can help you create new primers and also ensure the integrity of previously reported primers.

2. Order Multiple Primers

When ordering custom made primers for a particular gene, you can increase your odds of success by ordering 2 - 3 pairs of primers so in case if the first set does not give optimum results you can use others to continue optimization without hindering your workflow. This can be done, all the while keeping in mind your allocated grant funds, as primer manufacture is not a very costly process.

3. Make Sure DNA Sample is Pure

DNA isolation can be a tricky process especially if you are not using an extraction kit. But I always prefer manual methods for isolation that require diluting, preparing and sterilizing chemicals by yourself. I think it gives you a better feel for the procedure and is more cost effective. 

Once you have extracted the DNA, always run it through a 1% agarose gel to check for quality and impurities. A perfect extraction shows clear bands of genetic material but it wont travel far in to the gel as it has a high molecular mass.

1. If your DNA refuses to enter in to the well, it means that the DNA sample is contaminated with proteins and has not been purified correctly.
2. If the gel does not have somewhat clear bands and appears like a gradient then it means that the DNA has been damaged during the extraction process. 

Do not proceed if you encounter the above mentioned issues and redo the extraction procedure if possible.

4. Always use Controls

PCR is a process occuring at the molecular level so you cant exactly see what happens inside the PCR tube. For that you have controls and both positive and negative ones are important.

1. A negative control is a PCR tube with PCR complete PCR reagents minus the template DNA. This ensures that there was no impurity present in the reagents and the results that you obtain in the sample are true positives.
2. Positive control contains PCR reagents and a standard DNA template that will polymerize under the given conditions. A positive control means that the PCR reagents and the thermocycler are working properly.

5. Jumpstart your Optimization with a Gradient PCR

A gradient PCR is performed in a specialized thermocycler where each well can have different temperature settings. So you can set a different annealing temperature for your primers in each well and effectively eliminate negative results. Typically annealing temperatures range between 50℃ to 60℃. Be wary that while using reported primers, your optimized annealing temperatures can be different than those reported by other researchers.

6. Work on Your Pipetting Skills

Before actually starting off with PCR, take some time to learn how to effectively use a pipette. This will not only help you dispense accurate reagent quantities (mind you they are exceptionally low in volume) but also build up your pace. Timing is very important because if the reagents are not mixed with DNA and primers in the shortest time possible the reaction may not give results.

7. Play Around with the Taq Polymerase

Sometimes slightly increasing the units of taq polymerase can give you positive results if you cannot seem to have any bands despite adjusting the annealing temperature.

Increasing the amount of magnesium can enhance the activity of the polymerase too.

8. Always Prepare the Reaction Mix on an Ice Block

Taq polymerase is temperature sensitive, as soon as the temperature rises the polymerase activity begins. In order to prevent this reagents and DNA re always mixed atop an ice block.

9. Listen to Your Equipment

Carefully read manual of your thermocycler and ensure that it is placed in a well ventilated cool room, preferably placed in an air conditioned room. Each thermocycler is different and you get to know about its uniqueness while operating it.

10. Sequencing for the Final Verdict

If you want to be really sure that the amplified product you have is actually the gene that you had meant to amplify then the most straightforward method is to get it sequenced. Sequencing costs have considerably lowered so it would still fit into your budget if you sent representative samples.

11. Keep a Log Book

Before performing each optimization reactions, write up a log containing information about the annealing temperature, magnesium concentration and taq units. The log should contain dates and samples used. This will be hugely helpful in tracking your progress.