Hi all,
I'm trying to analyse bacterial diversity by amplifying 16S rRNA using primers targeting V3 and V4 region. The samples are human biopsy tissues.
After extracting the biopsy sample for total gDNA using the MoBio PowerSoil DNA extraction kit, the extracted DNA was used as PCR template, however I'm getting a very faint smear as the product migrates, what is causing this and has anyone else seen the same observation?
I've diluted my template and primer using EB buffer from QIAGEN. Does additional tris have an effect on PCR?
I'm also getting a larger and more intense smear for some prep, I suspect those are amplification when priming to the human origin?
The last 2 lanes from the right are positive control (Pseudomonas sp.) and negative control (10 mM Tris) respectively,
HI,
in the tested samples this looks like some unspecific products made by DNA polymerase. Try optimizing annealing temperature, change primers or polymerase to more specific. In the positive control sample the smear goes form the overload, so just dilute sample ~5-10 times.
Tris should not affect here, as lot of polymerases are working in tris buffers.
Hi Yu-fei. What you are seeing is quite a common phenomenon.
I notice from your gel there a tendency for the smear to be more prominent when there is less PCR product. This suggests that more template may help.
Also running for less cycles is often effective in minimizing non-specific products. Try reducing by 5 cycles. If the band intensity is then too low, increase template concentration.
Running the PCR with a higher annealing temperature maybe all that it requires. Try an extra 5oC.
Another method that could also help also is to use touchdown PCR, starting at an annealing temperature 5oC above what you are currently using, then coming down to your current annealing temperature.
Have you actually tested these PCR products in the downstream processes you are intending? Often sequencing works on PCR products that appear on a gel to be sub-optimal.
An alternative, if the above methods do not help, is to cut the bands out the gel, and extract the PCR products, so avoiding the non-specific products.
Best regards
Roger Latham
Thank you for your replies and suggestion.
I'm not too concerned about the non-specific product obtained and also because we are comparing this experiment with the previous. Ideally experimental condition have to be consistent.
Although temperature is definitely an issue, but is not to be address just yet. I was just wondering whether the faint white smear (from almost start of the well) reflects of problems in the amplification e.g.presence of inhibitors, quantity too low, complex sample... as such.
Increasing the template concentration is a possible option, however that means quantity of all the samples needs to be increased to reduce experimental bias. For those that have already obtained a intense band, non-specific amplification could then occur.
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