realtime PCR,meltcurves

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ammu
ammu's picture
realtime PCR,meltcurves

I am doing qPCR with SYBR green.i am getting standard curves almost right and the r^2  is 0.989 and the efficiency is 106.2.The problem is that for unknown samples ,i am not getting the good melting curves,some of the samples are having peaks below the threshold,i am doing the milk samples for DNA extraction.
So i want to know whether due to low quantity of DNA in the samples,i am not getting the good melt curves?
For standard curves,i used plasmid dna with the gene of interest.Please help me in this situation.Is it the problem of primers or samples?

Ivan Delgado
Ivan Delgado's picture
 

 
Hi ammu,

I need more information to help you out. What does it mean to have standard curves "almost right"? An r2 of 0.989 is ok, but that does not tell you much about the quality of your assay. You need to determine the slope, which should be between -3.1 and -3.6. 
When you say you are not getting good melting curves, what do you mean? Are the peaks too low or do you have multiple peaks? Are the peaks at the expected melting temperature or at a lower temperature, indicative of primer dimers? 
It is very important to validate a qPCR assay before using it. That is typically done by running a serial dilution of your template of interest (5 10-fold dilutions would be ideal). 
Last but not least, an efficiency of 106 is indicative of sample inhibition. Basically, it is likely that your DNA is not very clean. 

ammu
ammu's picture
Hi Evan,

Hi Evan,
Thanks for the reply.
The slope is -3.183.
For unknown samples,i am not getting good melting curves,ie i mean the peaks are too low,the peaks are at lower melting temperature and some are having multiple peaks as well.
I have validated the qPCR  by running with serial dilutions of template and now i need to compare or find out the copy numbers of organisms in the unknown samples with that of the standard curve.
I am doing 5 10 fold dilutions as well.
I am extracting DNA from infected milk samples which were found negative on conventional culture,so basically it will contain low quantity of bacteria and milk conatains PCR inhibitors also.
What you pointed out may be correct ,DNA may not be clean.
What should be the measurement on nanodrop for genomic DNA and A260/280?

Ivan Delgado
Ivan Delgado's picture
 

 
A slope of -3.183 is on the low end, but should be ok. The trick is that likely you obtained that slope using DNA that was not isolated in the same way as your milk samples. The ideal situation is that you need to validate your assay (run a standard curve) using DNA that was isolated almost the same way as the unknown DNA samples you intend to analyze. 

A melting curve peak that is too low could well mean that there is little to no signal present, so there is nothing wrong with that. Getting peaks at lower temperatures on the other hand is not good: it implies you have a non-specific assay or primers dimers. If that is the case, then you may have to re-design your assay. 

The readings you should be getting on a NanoDrop should be between 1.6 and 1.8 (260/280 ratio). If you get lower than that, it is likely that your DNA is not very pure.

ammu
ammu's picture
Hi Ivan,

Hi Ivan,
Thanks again.
Validating the assay using DNA samples isolated in the same way as unknown,i didn't understand it.
Also if the readings on Nanodrop is more than that,i am getting sometimes on unknown samples upto 2.028 like that(269/280)
I am taking only 1 microlitre of template,0.2microlitres of( universal primer)primer each(10pmol/microlitre)and 10microlitre of sybrmix and rest water..Is it ok?

Ivan Delgado
Ivan Delgado's picture
What I mean about validating

What I mean about validating your assay using DNA that was isolated using the same protocol as the one used to isolate your unknowns: if you use very clean DNA to validate your assay, then your assay is going to behave differently than when you run it using DNA that was isolated in a different way. By using DNA that was isolated using your standard protocol for validation, you are making sure that your validation reflects the way you regular testing will behave. You do not need to quantify your DNA for validation; all you need is take the DNA, dilute is a number of times, and run the standard curve. 

Readings of >2 for the 260/280 ratio can mean that you have RNA in your DNA samples. Did you RNase treat your DNA? If you are not sure it is always a good idea to run a gel to make sure your DNA is not only pure DNA, not degraded, and also does not contain contaminants.

Your reaction conditions sounds just fine. I would use more DNA to minimize pipetting error, but other than that you see to be fine. 

ammu
ammu's picture
H Ivan,

H Ivan,
Thanks very much.
I would do a standard curve with one of the DNA from milk samples that i have extracted.The concentration of DNA in my samples is between 8 to 10ng/microlitre
Earlier i used plasmid DNA with the gene of interest  for standard curve and compared it with unknown samples.
For plasmid DNA extraction, i used the RNase treated resuspension solution,but RNase was not used for the procedure for DNA extraction from milk samples.I don't know if any buffer of the kit contains that,and it was not mentioned to add RNase also,anyway i will check that.
So at what step i shall use the RNase?,For genomic DNA extraction, i am using the spin column method.

Also i will use more DNA as template,as you suggested to minimize pipetting error.

Ivan Delgado
Ivan Delgado's picture
 

 
Typically the RNAse step is performed before you pass your samples through the column, that way you get rid of the RNase enzyme and other related reagents before you get your purified DNA. 

ammu
ammu's picture
Hi Ivan,

Hi Ivan,
Thanks.I used universal primers for 16SrRNA and now i will validate my assay as you said.

sinharakesh
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At first in your case i found

At first in your case i found the problam of
1. DNA contamination
2 .Formation of primer dimmer
3.Unspecific binding of primer
4. Very low number or may be absent of target DNA

u can use Colum method for DNA extraction of  either Invitrogen or Qiagen for DNA where RNase treatment is either involved or some time you required to give. (See protocol for extraction).check the primer by computational method s about primer dimmer formation and other specificity G and C content
should be evenly distributed so no clump should be formed.

Check the same in PCR with different dilution if u got prominent single band in PCR than only proceed other wise change the primer or further reestablish all PCR condition. If possible tell me the size of amplicon you are getting in both the cases means in normal PCR and real time.