This month's question from the Molecular Biology Forums (online at molecularbiology.forums.biotechniques.com) comes from the “Real-Time qPCR/qRT-PCR Methods” section. Entries have been edited for concision and clarity. Mentions of specific products and manufacturers have been retained from the original posts, but do not represent endorsements by, or the opinions of, BioTechniques.Molecular Biology Techniques Q&A
Why doesn't my qPCR work with a high fidelity enzyme and SYBR green? (Thread 29684)
Q: I've always used SYBR Green or HYBProbe master mix kits for my qPCR reactions. But for my current project, I need to sequence the products following qPCR. My advisor says that a high fidelity enzyme should be used here since we will be sequencing the products, which means I can't use the master mixes.
I purchased 10,000× SYBR Green along with Phusion high fidelity polymerase and have been trying to use this combination for qPCR, but haven't been getting any amplification. The Phusion enzyme comes with its own 5× buffer that I add to my mix along with 0.2 mM of each dNTP, 0.5 µM of each primer, and 0.4 U polymerase in a 20 µl reaction. I use SYBR Green at a final concentration of 1×.
The primers work in qPCR with the SYBR Green master mix kit, and the Phusion enzyme works for standard PCR when the product is visualized on an agarose gel, so I assume the problem is coming from SYBR Green. I read that SYBR could inhibit PCR, but that it should be fine at an optimal concentration of 1×. I ran the qPCR products on an agarose gel and nothing is there, so it's not a failure in detecting amplification. Why isn't the product amplifying in my reaction?
A: I've read that there are proprietary fluorescence stabilizers in many commercial master mixes along with betaine, trehalose, BSA, glycerol, DMSO, formamide, KCl, pH 8.3 TRIS buffer, or several other additives. The amounts of these are largely held as trade secrets.
I assume you are using cDNA or DNA in your reactions so you don't have to worry about adding a reverse transcriptase enzyme. That would be the only big oversight I could imagine that might cause this problem. Since you are not using hydrolysis probes, you wouldn't have to worry about whether the Phusion enzyme has a 5′ nuclease function
You might try increasing your Mg++ concentration to 3 or 4 mM in your final 20 µL reactions. Some master mixes use 5.5 mM. This could increase the activity of your polymerase, but it might also decrease the specificity.
Your labeled oligos or primers may also have different effective Tm values in the self-made master mix, since I think Tm is partially dependent on the ionic strength and pH of the mix. If the Tm is different, you may require different thermocycling conditions. But even if this were the case, you should still have seen at least some amplification.
A: You can use the master mixes if you're planning to directly sequence the products. Even if you plan to clone, qPCR products are small, so you should only have to sequence a few colonies to find one with the correct sequence.
When adding SYBR Green, you usually need to dilute the stock about 1:40,000—1:50,000 for qPCR reactions. There are several published recipes out there (a couple in BioTechniques spring to mind). Try adding more Mg, up to about 3 mM, since this should help overcome inhibition by SYBR Green. If you're running the reactions in a capillary LightCycler, then you'll need BSA as well to prevent the reagents from adhering to the glass walls.
A: I agree that your master mix should be fine, although I don't know your specific needs. Regular Taq does have lower fidelity, but qPCR usually amplifies small products of less than 500 bp, so you're unlikely to encounter a mutation. As long as you confirm the same sequence in more than one clone, it should be okay.
If you sequence the PCR product, repeat sequencing won't be necessary either. When you sequence a PCR product, you are looking at thousands of sequences pooled together, so any mutations will be buried under many correct sequences. It's best to sequence both strands from both directions so any sequence dependent artifacts won't repeat.
Q: The SYBR Green I purchased came as 10,000× in DMSO. I diluted 2 µl of that into 198 µl of DMSO to make a 100× stock. From there, I added 5 µl to 95 µl of water to make a 5× aliquot for my PCR. I used 4 µl of the 5× in my 20 µl PCR for a final concentration of 1×. More recently, I tried using 2 µl of the 5× for a 0.5× concentration. I ran that reaction on my normal thermal cycler under the same conditions that consistently work with the Phusion enzyme, and two out of eight reactions showed low levels of amplification.
A previous post recommended a 1:40,000 dilution. I am starting with 10,000×, so 1× would be a 1:10,000 dilution, 0.5× would be a 1:20,0000 dilution, and 0.25× would be a 1:40,000 dilution. If I continued to make a 5× aliquot, I could add 1 µl per 20 µl reaction for 0.25× or 1:40,000 dilution? Is this correct?
Also, I make the 100× dilution in DMSO and the 5× aliquot in water. Is this the correct way to dilute it the stock solution? If I dilute to 5× in DMSO, it seems that would be too much in the final reaction.
I am using LightCycler capillaries, so I think I will need BSA as recommended. I found a reference using 5 µg BSA per 20 µl reaction. Does that sound like a reasonable concentration?
I will be directly sequencing the PCR product, which should be 153 bp.
A: I also make my own SYBR mixes using the 10,000× SYBR from Life Technologies. I think that your SYBR Green is too concentrated; I dilute the one hundred fold (1 µl in 99 µl H20) and use just 0.1 µl of this 1:100 dilution in a 50 µl qPCR reaction.
A: It sounds like the 10,000× SYBR solution is actually a 50,000× solution, 5× more concentrated than expected or advertised. Could it be that the 10,000× figure was originally based on an understood 100 µL qPCR reaction instead of a 20 µL reaction?
We're all familiar with the older, common suggestion of 50 µL size qPCR reactions. Over time, this was reduced to 25 µl and then to 20 µl to save money. 10-, 5-, and even 2-µl reactions on 1536-well plates are performed regularly now. Perhaps changing convention of reaction sizes has confused the definition of the original 10,000× SYBR Green solution?
The same thing seems to have happened with ROX solutions. Commercially available stock ROX solutions have to be diluted a lot more than expected before they reach the desired 30 to 50 nM final working concentrations suggested for many qPCR reactions.
I think that your SYBR Green solution should be empirically viewed as a 50,000× solution for dilutions, which should be done with water to avoid inhibiting reactions. Although DMSO can help qPCR reactions, it may already be in the master mix, so adding the extra DMSO as a SYBR diluent may cause problems.
DMSO at 2%-5% may be necessary for amplification of some templates, but 10% DMSO could reduce Taq or other DNA polymerase activities by up to 50%, so it should not be used routinely. DMSO is thought to reduce secondary structure and is particularly useful for GC-rich templates, but the benefits must be tested empirically in each situation.
DMSO at 5%, or about 0.7 M, is considered the maximum recommended amount for the final qPCR reaction. You are using nearly 2.8 M DMSO (4 µL/20 µL), which is way above the recommended concentration for PCR/qPCR enhancement. It looks like you are using DMSO at about 20% (or more if your master mix already contains DMSO) in your reactions.
You mentioned that you already saw better results at 1:40,000 SYBR dilution; 1:50,000 is only a stone's throw away. So, dilute your SYBR stock with water instead of DMSO and use a lower concentration of SYBR Green; hopefully that will remove the inhibition.
A: SYBR Green was developed as a gel stain, which is where the 1× calculation applies. That has nothing to do with qPCR. SYBR Green is known to inhibit PCR. One way to alleviate this is increasing Mg. I think you'll also need more BSA, probably 500 ng per µl if my memory serves correctly. An older paper by Ian Teo looked at these blocking agents for capillaries if you would like to look it up.
Q: You are right, the 10,000× SYBR I use for my qPCR is not actually sold for use in PCR; it is meant for use as a gel stain. I will make the adjustments recommended and dilute it down to a suitable qPCR concentration.