Chilling Bacterial Cultures Before Centrifugation (View forum version)



brightfield

Posted 07 February 2009 - 12:03 PM

We are having problems with MaxiPreps - the DNA appears to be degraded or at least, not supercoiled. One thing I noticed that my student does that I think may be a problem is she puts the overnight culture (in 250 mL bottles) directly into the 4C centrifuge without an initial "chilling" step. In my old lab, we always put the overnight cultures on ice for 30 minutes or so, to make sure they were cold before going into the centrifuge. Could this be the reason for our problems?

perneseblue

Posted 07 February 2009 - 06:36 PM

i don't believe so.

instead I believe the problem you are experiencing could be caused by
1 - too vigorous treatment (shaking) of the cell solution, once the cells are lysed with the lysis solution. This can shear the plasmid. The cell pellet has to be completely resuspended prior to addition of the lysis solution. And the two solutions mixed together gently.

2 - possible contamination of the reagents.

3 - using an expression strain rather than a cloning strain to prepare the plasmid.

TanyHark

Posted 07 February 2009 - 07:09 PM

We are having problems with MaxiPreps - the DNA appears to be degraded or at least, not supercoiled. One thing I noticed that my student does that I think may be a problem is she puts the overnight culture (in 250 mL bottles) directly into the 4C centrifuge without an initial "chilling" step. In my old lab, we always put the overnight cultures on ice for 30 minutes or so, to make sure they were cold before going into the centrifuge. Could this be the reason for our problems?

I always put o/n culture directly in 4C centrifuge. And I am the lab's maxi-queen :)

brightfield

Posted 08 February 2009 - 10:09 AM

We are having problems with MaxiPreps - the DNA appears to be degraded or at least, not supercoiled. One thing I noticed that my student does that I think may be a problem is she puts the overnight culture (in 250 mL bottles) directly into the 4C centrifuge without an initial "chilling" step. In my old lab, we always put the overnight cultures on ice for 30 minutes or so, to make sure they were cold before going into the centrifuge. Could this be the reason for our problems?

I always put o/n culture directly in 4C centrifuge. And I am the lab's maxi-queen :)


So, what other steps are critical or have you noticed that if you do something wrong leads to a "dirty" prep?