genecutl at mendel.berkeley.edu (gc) writes in article
Message-ID:<genecutl-080794200752 at kos2mac23.berkeley.edu>
>I've been using a boiling-lysis protocol for mini-preps and it used
>to work beautifully. The protocol involves vortexing the cells in
>STET buffer, boiling, spinning down the precipitated crap, then
>EtOH ppt the sup.
>The pellet I got from this has a huge amount of protein in it, but,
>surprisingly, my restriction digests always looked great and I even
>had much greater success sequencing this DNA than DNA from alkaline
>The problem is now the mini-prep gods no longer smile on me and I'm
>lucky to get a single sample to work out of twenty. What can have gone
>wrong? Please don't tell me I have to go back to phenol/chloroform
Unfortunately, all the "fast and simple" preps derive their speed and
simplicity from leaving out steps that make them reliable.
It's not clear from your description whether or not you have lost your yield
of DNA. If you don't know, run agarose gels on the template. If the yield is
variable, you may be vulnerable to carrying too many cells or too much culture
fluid into the lysis and diluting the lysis detergent. Also, consider that
there might not have been plasmid in the colonies in the first place, or that
you've gone to larger inserts or a different vector and are just getting less
plasmid to start with. A routine problem with mini preps is that the DNA is
fine but the quantitation is all wrong and so template/primer and
template/nucleotide ratios go astray. Vulnerability to this varies with the
sequencing method, and even the identity of the primer.
If the DNA is present but won't prime, then one usually assumes it is too dirty
with some diffusible inhibitor. It's worth checking this out by mixing the
problem template with a control template/primer. If it fails to inhibit
sequencing of the control template/ primer, then your problem is deeper in the
design of your experiment, not in the DNA prep.
These procedures can get in trouble if you increase the amount of EtOH, make it
a colder or longer precipitation, or spin harder. This will bring down both
more crud and more salt. If the DNA is dirty, sometimes you can dilute out the
problem by using less template in the sequencing reaction. Sometimes another
EtOH precipitation will clean it up. Another source of variability is that DNA
that hasn't been cleaned may degrade or precipitate upon storage.
Finally, if you are getting ladders, but the background underneath them is too
high, consider removing RNA.
Steve Hardies Hardies at thorin.uthscsa.edu
Please echo any followup to my mailbox; our news client is unreliable.