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.
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.
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