Hi,
I've run a PCR, with the DNA phusion KIT, to get a fragment of 125 bp, from FFPE DNA.
As I analyzed the results using our Fragment Analyzer, I noticed a very weak band in the NT control of the same size as the Positive Control, which of course led to the conclusion of contamination.
HOWEVER, in the lanes where I added template DNA, there is of course a strong band of the desired size, but in one lane where I added a very low concentration of DNA there is no band what so ever. A rough estimate is that the DNA concentration in PCR reaction with no band is 0.28 ng/µl.
I am a bit confused about this as I would have expected bands in ALL samples, due to the faint suspected contamination?
What could be the cause of this? Primer Dimers in purely empty samples?
•
written
by
Nicolai Skovbjerg Arildsen •
1 answer
Dear,
You mentioned that the size of the band in NT was the same as the size of the band at positive control. Than you mention, for other samples where you added the template DNA, you have a band of the desired size. Doesn't this mean that all the bands are the same? \
You mentioned primer dimers, depending on the concentration of your gel, you might not be able to separate 50bp (dimers) and 125 bp. Increase the concentration of your gel and re-run the PCR. Also, generally faint bands in NT are due to amplifying bacterial fragments in water. So if you want to make sure what you amplify is the correct region, you can always cut out the gel where the bands are and sequence it to make sure you have amplified what you wanted.
Lastly, try to increase the stringency of your pcr reaction. Decrease the MgCl concentration and increase your annealing temperature. You can also do a touch-down PCR, where you start with about 10 degrees higher annealing temperature and reduce it 0.5 degrees per cycle until you go down your original annealing temperature.
Regards,
Log in to answer this question.
-
NTC amplification close to expected amplicon size during primer validation – primer-dimer or non-sp…
written by Sashinika •
-
Tutorial: Premade library preparation – considerations, tips and tricks (II)
written by Novogene •
-
miseq double band
written by junghwachoi96 •
-
Real time pcr/qPCR primers specificity
written by muhindi_stephen •
-
Finding non-polymorphic region of genome for PCR primer design
written by joneill4x
-
How to merge pool-seq DNA samples
written by wang.yiguan •
-
PCR with 2 cycles gave no products
written by ramonaramonawan •
-
Gene set analyses using GSREG in R and interpretation
written by Nicolai Skovbjerg Arildsen •
-
Discrepancy between agarose gel and bioanalyzer after sonication for ChIP
written by augelloma •
-
What is a good threshold for calling variants in WES data
written by Nicolai Skovbjerg Arildsen •
Hello Nicolai Skovbjerg Arildsen!
We believe that this post does not fit the main topic of this site.
For this reason we have closed your question. This allows us to keep the site focused on the topics that the community can help with.
If you disagree please tell us why in a reply below, we'll be happy to talk about it.
A better choice would be [SeqAnswers][1]. SeqAnswers is a more general molecular biology/sequencing oriented forums, whereas BioStars is focused on Bioinformatics
Cheers!
If your negative control is a typical no-DNA control, then you have to throw the whole thing away. Don't waste time trying to guess which samples may or maynot be contaminated, just redo the PCR. Also, don't bother doing +ve controls if your fragments already have an expected size... you don't gain anything by having everything and your +ve control failing.
If it does turn out to be primer di/polymers, then you can't use that primer pair. Shitty primers have off-target effects and you will spend your life messing around with salt and temp gradients. Save yourself the hassle and redesign them :)