What is your template type? gDNA, pDNA, or else? Your DNA concentration seems to be too high 1ug/ul such high concentration may inhibit PCR reactions. try optimising the PCR with 10-50 fold dilution of the template.

SD

it is genomic dna of Pseudomonas

Hi.. 

it seems in first reaction you got band but failed when you repeated? if it is so, you have to think of -

1.if your template DNA got contamination with DNase - to check this, do gel electrophoresis with template DNA 

2. Primer contamination - one probability is primer contamination. To check that run PCR with same primers with another Template DNA (Pseudomonas)

3. amplification condition: please revisit your amplification condition (specially Tm).

by considering these factors , you will be able to rectify where is the problem and which step need to improve.

Hope this is helpful

Hi,

See the characteristics of your Taq, which is supposed to work quite well in a range of 1 to 100 ng for a volume of 25-50µL.

if you have inhibitors, adding BSA could solve the problem

DJ

Template concentration of 1.0 ug is high. So, you can take 0.5ul of template for 25 ul reaction. Include negative control in one of your reactions and check  without adding template, If you get amplication then probably your primers got contaminated with template which is giving false +ve. If your template DNA got degraded, try to extract fresh DNA and set up reaction using optimal levels of DNA as template.

Thank you for your response

As mentioned above by many, you need to include a positive control (even if you have not used in your first run) to make sure there is no problem with PCR reaction. We routinely use a whole colony of E.coli in 20ul reaction in colony PCR screening and never had any problem with templace conc., DNAase contamination. If you use right PCR conditions and correct reaction mixture, you should get the product even by using intact pseudomonas as the template.

Below is the question of one of the researcher on research gate. High template definitely hinders PCR reaction.

Why does high concentration of DNA templates obstruct PCR?

I have extracted Bacterial DNA from sea water, and I get around 165 µg/ml for the PCR. I used Promega PCR mixture, they suggested to use 50µg/ml of DNA template for the PCR. I tried to use 6x DNA template (2µl of DNA template) & I have no band. When I decreased my DNA template concentration (0.5 µl, approx 50-80ng) I got a band.

50ug is quite a lot of DNA for PCR. There are two possibilites: DNA binds magnesium ions to stabilize its own structure - the ions are essential for the Taq polymerase to function. This can effectivly hinder the reaction to work - this can also happen with too much primers or excess dNTPs in the solution.

The other possibility is the presence of PCR inhibitors from your DNA preparation. This happens frequently and is less of a problem with the lower DNA concentration you used in the end.

Why does high concentration of DNA templates obstruct PCR? - ResearchGate. Available from: https://www.researchgate.net/post/Why_does_high_concentration_of_DNA_templates_obstruct_PCR [accessed Aug 27, 2016].

Another facet to consider is that your primers bind to many sites in the total DNA isolated. All of these sites initiate a single strand of DNA in the first round. You only "get a PCR product" if both primers are able to initiate priming of the targeted site enough in further rounds to produce a product..

Too much target DNA, and the concentrations of:

one or both of the primers could be exhausted before enough copies of the target region is sufficiently amplified, or one or more of the dNTPs are exhausted before enough copies of the target region has been amplified.

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