Hi there,
In term of plasmid isolation, I tried to thrive DH5α host cell containing recombinant gene in three types of broth media which are LB, SOB, and SOC. All media added with appropriate volume of AMP.
After 16h later, The culture grown in SOB and SOC yields OD600 of 0.18 and 0.148 respectively. The culture, however, grown in LB has got value of 0.01.
I would therefore like to know whether or not it is advisable to purify plasmid from culture grown in SOB and/or SOC? If so, what strategy should I apply?
Has anyone experienced in such case?
Any suggestions will help much.
P.S. I used to perform plasmid purification with Mini Kit by QIAgene.
Well you can just give it a go.
Ot grow them longer in LB at 28degrees C, I had to do this for several difficult constructs.
Dear all
Thanks for your kind comments.
I have just tried to isolate the culture grown in SOC, and found that It yielded very, very low either concentration or purity. I assume that it was due to plasmid nature; I used pGEX which is relaxed plasmid (pBR322 ORI). To I know, relaxed plasmid could conform partially into stringent and, maybe, it affect the growth.
Does it make sense?
Patrick Drugga
Thanks for asking me.
If I take a look into Los et al. 2004 (attached) that one of indications of phage contamination is 'thick white foam on the culture (even when the culture is dense)' (pp. 118), it did not appear in any steps either in agar plate or culture in broth. So, I can say, no I do not have.
Here is the article
http://jag.igr.poznan.pl/2004-Volume-45/1/pdf/2004_Volume_45_1-111-120.pdf
Yes ,you can. You may separate the the native covalently closed circular plasmid DNA ( ccc ) form from the open circulat , linear forms.
Ref.Molnar J. et al APMIS suppleent 30, vol.100, p.24-31, 1992
The OD600 of all your cultures are very low, this will be the problem. Otherwise you can isolate plasmid DNA from all these cultures.
most of the kits prescribe use of LB media as it allows to grow bacterial to perfect OD600 ~2-4 - which ensures high plasmid yield and purity and its very hard to overgrow. TB and other rich media are fantastic but in many cases you'll be producing cultures with OD600>5 which will result in "overload" for many kits. just be careful with vol input
Naturally, you can purify your (probably multicopy ) plasmids from nutrient broth. In agarose gel elfo you may check tthe three different formats of plamid DNA as CCC, Oc and Linear forms. I wish you many success.
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