Hi everyone!
We’re performing a bead-based size selection of double digested genomic DNA, targeting fragments between 400bp and 700bp, but we’re having some trouble getting rid of larger fragments. Our size selection protocol employs Serapure beads, prepared following the protocol in Faircloth and Glenn (2011), attached.
To test the efficiency of the Serapure in removing the larger fragments, we size selected DNA ladder using different proportions of Serapure (0,55x and 0,65x of DNA volume). Here is the electrophoresis gel of both the DNA remaining in the supernatant and eluted from the beads. Theoretically, we should see the larger fragments in the beads (marked as 2a, 2b, and 4a, 4b), and only the smaller fragments in the supernatant (marked as 1a, 1b, and 3a, 3b). Unfortunately, not all of the larger fragments seem to be binding to the beads and are still visible in the supernatant (although in a lower concentration, considering that the bands are not that bright).
One of our thoughts is that we’re not using the right proportion of PEG. The amount of PEG8000 used in the Serapure preparation is the same as described in the protocol (9g of PEG8000 and 1mL of Sera-mag SpeedBeads to prepare a 50mL Serapure solution).
I read some very helpful threads about bead-size selection, but still couldn't figure out what seems to be the problem with our experiments.
Any help is very welcome. Many thanks in advance.
-c.
We’re performing a bead-based size selection of double digested genomic DNA, targeting fragments between 400bp and 700bp, but we’re having some trouble getting rid of larger fragments. Our size selection protocol employs Serapure beads, prepared following the protocol in Faircloth and Glenn (2011), attached.
To test the efficiency of the Serapure in removing the larger fragments, we size selected DNA ladder using different proportions of Serapure (0,55x and 0,65x of DNA volume). Here is the electrophoresis gel of both the DNA remaining in the supernatant and eluted from the beads. Theoretically, we should see the larger fragments in the beads (marked as 2a, 2b, and 4a, 4b), and only the smaller fragments in the supernatant (marked as 1a, 1b, and 3a, 3b). Unfortunately, not all of the larger fragments seem to be binding to the beads and are still visible in the supernatant (although in a lower concentration, considering that the bands are not that bright).
One of our thoughts is that we’re not using the right proportion of PEG. The amount of PEG8000 used in the Serapure preparation is the same as described in the protocol (9g of PEG8000 and 1mL of Sera-mag SpeedBeads to prepare a 50mL Serapure solution).
I read some very helpful threads about bead-size selection, but still couldn't figure out what seems to be the problem with our experiments.
Any help is very welcome. Many thanks in advance.
-c.
Comment