Remember that "increase" and "decrease" actually refers to the final concentration of the components, and not necessarily the volumes.

Most commercial PCR mixes have a recommended final concentration since the manufacturer already optimized the buffer, enzyme, salts, etc. If you really want, you can use slightly less by replacing some with water, but it's normally not worth the bother.

If your primers are too concentrated, you can fail to amplify your desired product. Too dilute and you will also fail to amplify.

The concentration of cDNA (or other template) can be increased or decreased depending on the abundance of your target sequence. For example, if your target sequence is highly expressed, then you can start with less cDNA. If it's a low-abundance transcript, then you may need to increase the starting concentration.

To be honest, most scientists do NOT adjust the concentration of the Master Mix ever. They might increase or decrease the total reaction volume, increase or decrease the amount of template, or add in DMSO/Mg2+/etc. The only adjust the concentration of primers if they have tried other solutions and not seen any success. Most PCR optimization is finding the ideal annealing temperature and/or extension times for different sets of primers and choosing ones that work the best. PCR is a tool that we use to make constructs and answer questions. There is no need to fuss around with a protocol that works.

Good luck!

Dear Hamza Sohail

Very interesting question.

The question what you asked is what pcr optimization is:

1. Usually, if you increase primer concentration, you may get hazy product in gel due to decrease in specificity (more primers having chance to bind unspecific sites) or cause primer dimers to form (primer having more chances to bind each other).

2. If you increase cDNA, you have more chance to detect your target gene but as cDNA has carry-over products from the reverse transcription process, there may be inhibitory agents and inhibit your PCR. The MIQE guideline says dont use more than 10% of reaction volume to decrease the inhibition.

https://www.gene-quantification.de/miqe-bustin-et-al-clin-chem-2009.pdf

3. If you increase the mastermix or PCR buffer you may waste your expensive reagents or the Taq Polymerase may not work in increased buffer concentration.

While slight increase or decrease in these componenets dont impact your result, it is better to see what concentrations of each components (primers, MgCl2, cDNA, etc.) is giving the best results. This is called optimization and you can find how to do optimizations if you search in the web.

Divya

I just want to add to Divya's answer that at increased concentration of Taq I have observed increase in non specific amplifications (along with the targeted fragment).

Dilkaran

Remember that "increase" and "decrease" actually refers to the final concentration of the components, and not necessarily the volumes.

Most commercial PCR mixes have a recommended final concentration since the manufacturer already optimized the buffer, enzyme, salts, etc. If you really want, you can use slightly less by replacing some with water, but it's normally not worth the bother.

If your primers are too concentrated, you can fail to amplify your desired product. Too dilute and you will also fail to amplify.

The concentration of cDNA (or other template) can be increased or decreased depending on the abundance of your target sequence. For example, if your target sequence is highly expressed, then you can start with less cDNA. If it's a low-abundance transcript, then you may need to increase the starting concentration.

To be honest, most scientists do NOT adjust the concentration of the Master Mix ever. They might increase or decrease the total reaction volume, increase or decrease the amount of template, or add in DMSO/Mg2+/etc. The only adjust the concentration of primers if they have tried other solutions and not seen any success. Most PCR optimization is finding the ideal annealing temperature and/or extension times for different sets of primers and choosing ones that work the best. PCR is a tool that we use to make constructs and answer questions. There is no need to fuss around with a protocol that works.

Good luck!

Hi Hamza,

Katie gave you a good answer.

You usually start by trying a standard reaction as suggested by manifacturer and then, if you do not obtain a result 100% satisfying, you can try to optimise..

This means changing some concentrations to improve for example the yield in specific product, or to make the reaction more specific, getting rid of aspecific products.

For example, all master mixes contain divalent cations, such as Mg2+ or Ca2+. these are critical cofactors for DNA polymerases. to increase fidelity of replication you must have low concentrations of these divalent cations, if you want to increase yield of a product in a reaction that gives you only the desired product, you may add some more Ca2+ that that present in the standard buffer.

If your primers are too concentrated, you may get dimers or aspecific amplifications, whereas too little indeed will result in little or no desired product...

cDNA should always represent no more than 1/10 of the reaction volume, because it can have inhibitory effects on PCR. Of course, below this top limit, as Katie said, you can try different dilutions to get an optimal amplification (abundant desired PCR product and no aspecific bands).

If your target is GC rich, you may improve amplification by using some DMSO...

Of course, particularly in standard endpoint pcr, you may also work on lowering or increasing the annealing temperature of your amplification, increasing the cycles (remembering that fidelity requires that you use the least possible cycles, as in late cycles, when reagents are running out, the error rate increases).

So, it really depends on what type of PCR and what is the aim of your amplification... But you usually start from standard conditions (annealing temp 2-3ºC below the theoretical calculation) and then you adjust conditions if needed.

Good luck!

Katie A Burnette Divya Rana Dilkaran Singh Paola Campomenosi Thanks to all of you for your valuable comments

Good answer from Katie A Burnette