Hi there,

I usually use a few hundred ng of genomic DNA for 50µL reaction with Phusion polymerase (ideally 250 ng according to the leaflet). DNA is very soluble in water then you don't have to wait too long after addition. Except if the DNA pellet has been overdried, in that case you'd better let it rehydrate for a while...

you can DNA dissolve by 50 ul from water and can use DNA dissolve direct in method or keeping. In PCR using 5 ul from DNA extract.

Hello Ahmed,

I use between 100 and 200 ng of genomic DNA (also extracted with Promega kit) for PCR amplification.

Good luck

Hadjkasem Basma , how many ul do you dissolve dna in? How many ul fo u use for pcr.

Thanks all for your help

I use 100 ng of DNA for 50 ul of PCR. 

I use 100 ng of isolated DNA from PBMC by trizol or QIAamp DNA in a 25 ul final PCR reaction 

Hii Ahmed, first calculate the concentration of the dissolved DNA spectrophotometrically. Then use it for PCR. Generally concentrations between 50-200 nano grams of DNA will do for most of PCR reactions. It is dependent on amount of DNA you add, not the volume. Even if you add very less volume of highly concentrated DNA, it can result in PCR inhibition/unwanted results.

Hit and trial is the best scientific approach if you don't know exact concentration of your protein. 50-100 ng should be fine or 1-2ul in 50 ul. Good luck

I use 200ng DNA isolated with QIAmp DNA Kit

For standard PCR amplification I use 50-60 ng of genomic DNA extracted from blood with Qiagen kit in 50 ul PCR reaction. it would be better to determine your genomic DNA concentration.

Hi,

 As you have eluted in minor quantity, then your eluted DNA would be much concentrated. You can dilute your DNA in 1:10 dilution and from the diluted DNA you shall use 1  Microlitre. That is 100 ng of DNA for a 25 microlitre PCR reaction.

I agree with above answers. we use 5 microliter of DNA template for 25 microliter reation. so follow kit protocol. calculate DNA conc and then decide how much to add.

regards,

Dear,

According to extraction kits, concentration of DNA differs. In general PCR reaction needs at least 30 ng, whereas in case of promega, you can use 3 ul. This is will be enough.

If you have a very concentrated dilution, its better to live the dna with water some hours before use for PCR, to help the mixing

In my opinion, for a PCR reaction in 25ul you only need about 20-40ng of DNA (depending on DNA quality). 

we usually dilute DNA to 25ng/µl and we load 4 µl (100ng) for PCR reaction, it works very well.

In my opinion, you could use between 50-100ng, depending on the initial concentration you have. For a 50 uL reaction, you could use 5-10uL as much. If you have less concentration reaction could work with less DNA (20ng for example) but it's not the optimal concentration. It depends on the quality as well, not only the quantity.

I recommend resuspend the dna in water al least 5 min and then mix for use for pcr reaction, for to know how quantity dna use I recommend check your dna sample with electrophoresis using standard dna ladder for quantification, you can see integration and quantity of dna. then when you to know the dna concentration you can use about 50-100 ng per pcr reaction. lucky¡¡¡

Dear,

I use in a PCR reaction in a final volume of 50uL, 50-60 ng of genomic DNA (depending on DNA quality)

Current methods (for example, Direct Blood PCR Kit from Thermo Fisher) allows to make PCR directly from the blood without extraction. You might be interesting in that.

http://www.thermoscientificbio.com/pcr-enzymes-master-mixes-and-reagents/phusion-blood-direct-pcr-kit/

For PCR amplification use 5 micro liter of DNA, having concentration of 50-60 nano gram. So before using DNA check out concentration of your DNA. 

Thanks a lot for your precious answers