need some help..covaris and dilutions

[seqgirl123]

Can someone please confirm I did below correctly. I am trying to figure out why my libraries aren't giving a high quant after PCR cleanup, so I'm first seeing if there is something wrong in the way I calculated the DNA concentration that was sheared on the Covaris, maybe there something about the Covaris technicalities I am missing. I have 2 dilution cases here if someone can correct them.

1. I have a DNA sample stock of 1ug/ul and want to make up a dilution of 5ug of it in 200ul. So using C1V1 = C2V2 formula, I did
C1 = 1ug/ul
V1 = x
C2 = 5ug in 200ul = 25ng/ul = 0.025ug/ul
V2 = 200ul

V1 = C2V2/C1
V1 = (0.025ng/ul)*(200ul) / 1ug/ul

V1 = 5ul of 1ug/ul DNA stock + 195ul of buffer gives 200ul of a diluted 5ug stock

I take 100ul of this and shear on the Covaris, while the other 100ul is kept as stock. So I would be shearing an aliquot of the 5ug stock. I am pretty sure this is correct, but would be nice if someone can check.

2. I have another DNA of 50ng/ul and want to make up 3ug of it in 200ul.
C1 = 50ng/ul
V1 = x
C2 = 3ug in 200ul = 15ng/ul
V2 = 200ul
V1 = (15ng/ul)*(200ul)/50ng/ul

V1 = 60ul of 50ng/ul DNA stock + 140ul of buffer gives 200ul of a diluted 3ug stock. I take 100ul of this and shear on the Covaris, while the other 100ul is kept as stock again. So I would be shearing an aliquot of the 3ug stock.

[ECO]

Your math is fine.

What step quantitation are you referring to? Immediately post shear, or at the Ed of he process? I assume these are Illumina libraries? How many cycles of PCR did you do? Did you check your yield prior to PCR?

[seqgirl123]

Yes these are illumina libraries. I don't check yield prior to PCR, because I always thought the yield prior to PCR will be low anyway. Is there a range of concentration I should be looking for though if I check?

My other question is if I did the math another way to dilute #2. I change V2 from 200ul to 50ul, so I will make up only a 50ul final volume, not 200ul. My doubt is whether this way really gives 3ug in the end or 750ng final concentration:

C1 = 50ng/ul
V1 = x
C2 = 3ug in 200ul = 15ng/ul
V2 = 50ul
V1 = (15ng/ul)*(50ul)/50ng/ul

V1 = 15ul of 50ng/ul DNA stock + 35ul of buffer gives 50ul and I use the entire thing for shearing.

[cldashie]

Hi seqgirl123,

Starting with 1 or 2ug of DNA should be plenty to check. You really shouldn't be losing too much DNA along the way, we seem to lose the most at the gel extraction. Are you able to see a band on your gel before the pcr amplification?

Also, on our Covaris the protocol suggests that your sample be in 120ul. If you have an air bubble in the tube I believe that it can affect shearing. I would suggest running a ul on the BioAnalyzer if you have access to one after shearing to see how your sample looks.

[Hamid]

Hi,

Your math is fine, but remember that if you are making making a 200ul solution with 5ug of DNA, and then using 100ul for fragmenting on the Covaris, you are actually fragmenting 2.5ug of total DNA. for your 3ug/200ul sample, you are actually shearing 1.5ug of total DNA.
Also please make sure that you are fragmenting at 130ul volume since using lower volume might cause the formation of an air-gaps which can increase the size distribution.

Thank you

Hamid