I am cloning different plasmids using normal cloning protocol.
Then I pick colonies of this cloning and run a mini-prep.
DNA of the mini is always subjected to double digest in order to check for the right cloned product.
After that I take 10 micro-liters from the right mini bacterial culture and inoculate on agar plate to get the single colony which I use to do a maxi-prep.
Last couple of time I did maxi, I got so low Conc. of maxi-DNA which upon sequencing give unreadable signal. I use the same Invitrogen kit without changing anything which used to yield a high conc. of DNA.
What may be the reason behind such disturbing problem?
Has this issue arisen with the same or with different inserts and/or plasmid backbones? What antibiotic are you using for selection?
Dear Alejandro,
Thanks a lot for your concern.
This happened with three different plasmids.
I am using Ampicillin.
Well, there could be a few things you might need to consider. One is the bacterial. selection pressure as it plays an important role in its growth. Plasmid yields will go down without the selective pressure to keep it.Secondly, OD600 can adversely affect your isolation with a high OD600, you overwhelm the chemistry of the kit while, Iow OD600 there won’t be a lot of bacteria to get your plasmid from.
While, If the culture conditions are not the problem,it can generally be due to Solution 2 (one containing NaOH and SDS) or the quality of isopropanol.
Hopefully this helps.
Cheers!
confirm OD600 for your culture- by Nanodrop or any other spectrophotometer. It should be in the range 2-4 for perfect prep. Then use a robust maxi plasmid prep kit, such as PureLink HiPure or PureLink Expi endotoxin free, with 100 ml LB culture input for high copy plasmid
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