How to choose right volume of DNA and SYBR Green

Question

I'm testing fluorescence level of a sample having dsDNA, SYBR Green and milli q water but I'm facing difficulty in choosing right volume of dsDNA and cons level of SYBR Green.

Details of sample: dsDNA:1000 nano mole, SYBR Green:1X (1000 times diluted from original stock)

Sample_1: dsDNA (3ul) + SYBR Green (5ul) + milli q water (27ul)

Sample_2: dsDNA (3ul) + SYBR Green (5ul)

Result: In first sample I haven't got any signal but in second case I got( But I want 5 times higher then what I got).

Please tell me how it work and how these things depends on each other. I'm not a biologist if possible suggest me resource as well.

Answer 1

I had to look it up online, but I eventually found here an important information: the detection limits of SYBR Green.

dsDNA at 300nm transillumination: 60 pg
Oligonucleotides at 300nm transillumination 1-2 ng

You should first check the size of your DNA of interest, and from its size figure out its approximate molecular weight (for example using this online calculator). Then, you should check that 1000 nmoles of your DNA corresponds to a mass greater than 60 pg. If not, you should increase the amount of DNA in your assay accordingly until the mass introduced in the assay exceeds the detection limit of SYBR Green.

Now, do you know the concentration of your DNA? In other words, how did you choose to use 3 uL of DNA? How do you know that these 3 uL contain enough material? (1000 nmoles as in your protocol, or better, that this is higher than the detection limit in terms of mass).

Also, did you use the 1000X stock solution of SYBR Green without intermediate dilution? If so, the final concentration of SYBR Green is much larger than the final 1X you want, in both of your samples:

1. sample 1: 5 uL of 1000X SYBR Green in a total volume of 3 + 5 + 27 = 35 uL means that the final concentration is 1000X * 5 / 35 = ~143X.
2. sample 2: 5 uL of 1000X SYBR Green in a total volume of 3 + 5 = 8 uL means that the final concentration is 1000X * 5 / 8 = 625X.

What you need is either use 1 uL of 1000X stock solution of SYBR Green + whatever amount of DNA that will be above the detection limit + buffer to adjust the volume to 1000 uL final (which will bring the SYBR Green to 1X final), or make an appropriate intermediate dilution of SYBR Green so that its final concentration is 1X by following the sample preparation as described in your question.

The sample preparation you describe here yields a composition that is way off the one suggested by your protocol, at least for the SYBR Green. Until you try again with proper final concentrations, do not conclude that it didn't work. :-)

Good luck with your project.