Genomic DNA extraction - viscosity? - (May/15/2007 )
I did a genomic DNA extraction of ES cells in a 96-well plate. I used 50uL of a sarcosyl-based lysis buffer, 40deg C overnight. Then added 100uL ice cold EtOH at room temp overnight (as a 96-well plate spinner was unavailable). I poured it off, washed 3x with 150uL 70% EtOH and dried (without overdrying). Dissolved in 25uL 0.1x TE and used that for PCR.
The issue is that the final solution was so viscous that I am wondering if that could cause a problem in PCR? I have not done this in a long time so I am curious if it is always supposed to be this viscous or whether I should use more 0.1x TE to dilute? I was only able to even pipet 1uL of it by mixing the well thoroughly and repeatly pipetting up and down until enough came to fill up 1uL of the pipet tip, otherwise I couldn't even pipet it. Plus when I put it into the 96-well PCR trays, it would stick to the sides and not run down to the PCR reaction buffer. I assumed when the solution was boiled for hot start and during the PCR water condensation would mix it up and it appeared so after PCR.
Does anyone have any more experienced advice as to the the proper final consistency or if that will affect the PCR reaction at all? I did get PCR products in maybe about half the lanes (did the whole 96-well plate), though I didn't expect to see them all anyway for the first PCR reaction. I should see a product if neo gene still in there for one set of PCR reactions. Then the other 96 reactions were primers overlapping the whole gene (giving 2 different sized products depending on if neo cassette still in there) so I should see product in every lane. The other issue could be since the large product (2nd PCR rsn) was so large, ~2kb, maybe it was too long. We did extend the extension time, though.
Thanks for any input.
there is something wrong here, a DNA solution should not be that viscous. it is probably contaminated by polysaccaride/cell debrie from the ES cells.
However as you are getting a signal in half the wells, this is not a terminal problem. THere is not much I can recomend, as the samples are in a 96 well format.
If you do have a centrifuge that can take the 96 well plate and a multichannel pipette. You could try spining the samples after the overnight incubation with the sarcosyl-based lysis buffer. Once all the junk is pelleted, the supernatant containing the DNA can be pipetted to a new 96 well plate. Then followed by EtOH percipitation.
Resuspending in a large volume of TE, will help with pipetting the templates.
If your signal is clean, increasing the magnesium ion concentration can help. Try increasing it by 50% to 100%.
Do you get rid of proteins in your procedure ? this may help already, but generally gDNA is pretty viscous anytime.
well i noticed that when my DNA preps were too concentrate, they were viscous.
it's not all time, but tend o be.
Second question is : why use 0.1X TE ?
You may dissolve in 10mM tris hcl ph8 if you're afraid about EDTA rather than your 1mM. should enhance.
You may have a RNA contamination. i'm not too sure if this point is relevant with sarcosyl, but in midipreps by alkaline lysis, the RNA gelify strongly. if you think it may be useful, RNase treat your sample and prot K after.
finally, there may be proteins remaining.