Preference in isolation of plasmid DNA methods? - (Aug/02/2007 )
I bet you guys out there had experienced the problematic step (or none at all?) in isolating plasmids for TRANSFECTION, RESTRICTION STEPS, CLONING etc... Sometimes you get a fat piece of band in the agarose gel, sometimes faint and at times zero, zilch?
I've had my share. Sometimes I get lots of plasmid and sometimes, very little. I'm using Qiagen and Invitrogen kit and both had not been giving consistent results. I was wondering if there're other proven methods in your lab that you've used to isolate plasmid with a happy face shown each time you measure the concentration of the plasmid yield? Would appreciate it very very much if you could share it.
Oh ya. Anyone using Sambrook's alkaline lysis for plasmid prep and later for transfection?
For doing stock plasmid, or for restriction / cloning use, i do alkaline lysis method, the one including PEG/NaCl overnight.
Fr transfection assay, or in vitro transcription, i prepare my plasmids by quiagen columns. The yield is quite bad (my routine was alkaline lysis for so long that i belived many times i loose all the plasmid. In fact the very small pellet i saw was it ).
Yields for 50ml culture :
roughly 400µg for alkaline lysis method
Routinely 100-150µg with column and plasmids of 6-7 kpb.
In our lab, Qiagen and invitrogen gives us pretty consistent results but it depends on the plasmid. Some plasmids we get high yield and some poor. For many of our vectors, we do a day culture and use it to inoculate the overnight culture and this ends up giving atleast 500ug if the maxi prep was not good. Else we get between 1.5 - 2mg of DNA from a single column using Invitrogen kit.
if we do not use the day culture, we get less than 100ug, may be even 50ug from the same kit.
In my lab we use Alkaline lysis to prepare all our DNA; plasmids, BAC and PAC for cloning, transfection (yeast and mamalian cell) and microinjection (the DNA goes through a Qiagen gel extraction column to clean it further... ;p ).
Our experience with kits hasn't been good. Too expensive, with poor yeilds (compared to alkaline lysis). I believe the amount of DNA present in the lysis, exceeds the columns binding capacity.
Thanks Fred, Scolix and perneseblue.
I'll try on Scolix suggestion to use "day culture", and use the alkaline lysis step to get the plasmid. If the purity's not ok, can combine Qiagen's purification ya?
i use alkaline lysis method and i further purify the DNA with Cesium chloride gradient. yea it takes a long time to get pure DNA in this way, but i used to do it becuase using this method surely i got 3-5 ug/ul pure DNA.