I suggest you, first check your detection limit of your Primers using templet gradient PCR. With this experiments you will know the limit of your Primers.

Check your extracted DNA over gel to check DNA quality and intect or not. If your DNA is Pure DNA not an total DNA than 1ng/ microliter is also more than enough for PCR. First check primers detection limit.

In PCR reactions should be at least 10 ng of DNA.

20 ng is good.

What is the matter?

• Agreed
• 20ng to 50ng is usually optimal for decent purity DNA and decent primers
• Accordingly, you could try say 5ul; but the amount suggested all other things being equal should be fine
• A minimum of 10ng as already suggested so 2-3ul should be fine

Hi,

this depends on several factors including the used chemistry, detection method and assay. qPCR can cope with up to 100 ng depending on the detection system (Sybr Green based analysis may give you issues as the huge amount of starting DNA is already generating a fluorescence signal).

For End point PCR you can also try to add higher concentrations if you desire to have a very low LoD.

Best Sven

PCR PROTOCOL:

Initial Denaturation PCR activation: 95 Degrees Celsius for 5 mins

40 Cycles of:

Denaturation: 95 Degrees Celsius for 30 seconds

Annealing: 58 Degrees Celsius for 30 seconds

Extension: 72 Degrees Celsius for 30 seconds

Further Extension: 72 Degrees Celsius for 10 mins

Infinite Hold: 4 Degrees Celsius

I suggest you, first check your detection limit of your Primers using templet gradient PCR. With this experiments you will know the limit of your Primers.

Check your extracted DNA over gel to check DNA quality and intect or not. If your DNA is Pure DNA not an total DNA than 1ng/ microliter is also more than enough for PCR. First check primers detection limit.

Hello Vasilli,

As others have stated above you should be fine anywhere between 10-100ng of total genomic DNA, given it is of decent quality and you hvae good primers. I, personally prefer using 50-100ng if I have enough DNA.

So it's really a question of how much volume of your 5ng/µl stock you have, i.e. how precious the sample is.

If you have a lot of volume just use 5-10µl. If not, the 3µl that have been suggested to you should work as well.

Scaling up your total PCR reaction volume shouldn't be a problem - keep everything as it is, just adjust the buffer and top up with H2O, say to 30 or 50µl.

Your PCR prot. looks OK, although you'll probably get a similar result with 10 cycles less.

Annealing temp as well as extension time depend on your specific primers and amplicon length, respectively. If this has worked before just keep it like that.

Hope that helps!

If you don't pass the amount of 200 ng of DNA, and work with 10 ng or more you shouldn't have any problem at all. Good luck

PCR reactions should be at least 10 ng of DNA 100 ng is good.

As it seems from the Figure the resolution of DNA marker is good, but you have somehow problem with your running buffer so check your running buffer and reduce the voltage of electric current during running. The second thing, put double of the DNA quantity that you have used in the previous reaction. I think you will get good resolution of PCR products when you optimized your handling.

Good wishes

Dear Vasillo

Greetings and thank you for your question

As I see the work have some band and missing ones so try do PCR one again and be sure that the missing one have the same concentration of DNA by checking the concentration and try deliute it in 1:5 or other ratio

Having looked more closely at the gel itself I think Kamal might actually be onto something

The only thing that might point to a sample specific problem is the fact that the markers- although distorted- are still actually discrete

Nevertheless in conjunction with sample specific advice keep your options open and follow Kamals advice about the gel As well:make fresh running buffer; make fresh agarose with it and try not to run your gel above 150v and especially not 200v

I have seen that sort of smearing and distorted albeit discrete markers with gels that have too much current pumped through them with excessive voltage. Reused agarose or fresh agarose made with old precipitating buffer would exacerbate that problem. That at least would lead potentially to less distorted markers

You have right dear professor Laurence Stuart Dawkins-Hall, but I would prefer not to raise the voltage over 100 v.

Regards,

The voltage (recommended maximum) would depend on the size of the gel. So, smaller or larger agarose gels would have different limits. The gel size actually refers to the distance between the two electrodes, and recommended maximum 5 volts per centimeter.

i recommend toy use a positive control of viral DNA alongside your specimens to reduce your expectation. once you get a positive results of this control you have to realize that your specimens don't have any viral DNA. But when your positive control didn't give you any results, you have to look at the purity of your extracted template.

Are you sure the purity of you template DNA is 1.8?

if no, try to repeat your DNA extraction and assess the purity of your extracted DNA before its being submitted to PCR.

if yes, you have to reconsider the specific primers used for PCR. in the attache fig. all the electrophoresis conditions used were optimum as your PCR ladder was acted very well.

regarding the concentration of the template, 2 microlitter (of pure gDNA) is nicely suffice your demand.

First of all you must see the band of DNA you extract in 1%agarose so clear and not damage