QIagen maxi prep - urgent....please help (Aug/19/2005 )
has anyone used the qiagen maxiprep kit before? this is my 1st time going to use it and i was wondering if i could use a 2week old e.coli culture grown in LB broth and stored at 4 degrees in order to grow up 100ml of culture for maxipreping? is it ok? what do u all think? any experience anybody? can i use an old culture to inoculate and grow up a larger volume of culture for maxi preping and wud i be successful in getting high copy plasmids? urgent....please help
What antibiotic are you using? I know of several people using ampicillin who lost their plasmid along the way. Mostly I believe because the unstabillity of amp and also because ampicillin-resistant e. coli will convert ampicillin (so its concentration decreases rapidly in culture, and even when stored @ 4°C). So, what I would do is try a small culture volume first, if you still have the plasmid, go for the maxiprep.
Don't do it. If you need to recover the sample, plate some out on an amp plate, repick the colony, and grow more cells up from the colony. If you have a frozen stocik, start with that instead.
Most of your cells are likely dead, and dead cells chew up things like DNA.
If you need to store grown cultures for prep for long periods, then you can freeze them in the -80 after spinning them down and decanting culture medium. This has little effect on the plasmid isolation.
Most of your cells are likely dead, and dead cells chew up things like DNA.
If you need to store grown cultures for prep for long periods, then you can freeze them in the -80 after spinning them down and decanting culture medium. This has little effect on the plasmid isolation.
hi guys,
thanks a lot for your reply. i guess i'll use my frozen stock and plate on a new agar plate to pick colonies and re-grow them. but.....i think i have another problem. ....my frozen stock was made using an 1.5 weeks old culture in lb broth stored at 4 degrees. of course, i didn't directly use that old culture. what i did was, i took a few ul from the old culture and inoculated in a tube with fresh lb broth...and then i shaked the tube overnight at 37 degrees. and then, the next day, i created a ffrozen stock using this new tube...so my question now is, do u think my frozen stock is ok...or have i lost my plasmids? pls help...
If it is growing in selective medium and on selective plates, then there is at least some resistance gene being expressed. Likely it is the one you are using in your plasmid, but there could have been rearrangements -- unlikely, but certainly possible.