H*g 发帖数: 2333 | 1 Sorry only could type in English using lab computers
I a trying to express protein in E.coli and am wondering if I should save
glycerol stocks for transformed
BL21(DE3) or Rosetta2(DE3) strains for future or should we always start from
a new transformant?
How often does mutation occur in these strains?
Thanks a lot! |
n***w 发帖数: 2405 | 2 I do glycerol stocks and expressed my protein for couples of times and at
this point, no mutations found. And honestly I do not know the answer... |
T**********t 发帖数: 1604 | 3 Of course make glycerol stocks for those strains.
It's usually recommended to renew those stocks every 6 months. But I've used
frozen stocks of several years old and still got satisfactory expression. I
guess it may vary from case to case, though.
I would do small scale expression test each time before a large scale
expression culture if I'm worried about the quality of the frozen stock.
Just use a 5-ml culture and induce as how you would with a 1-L or larger
culture (scale down the amount of IPTG, of course). Then lyse the cells (
before and after induction) and run lysate on a SDS-PAGE gel to check
expression level. |
n***w 发帖数: 2405 | 4 Actually many protocols say high concentrations of IPTG can be used at
screening stage, up to 1mM.
used
I
【在 T**********t 的大作中提到】 : Of course make glycerol stocks for those strains. : It's usually recommended to renew those stocks every 6 months. But I've used : frozen stocks of several years old and still got satisfactory expression. I : guess it may vary from case to case, though. : I would do small scale expression test each time before a large scale : expression culture if I'm worried about the quality of the frozen stock. : Just use a 5-ml culture and induce as how you would with a 1-L or larger : culture (scale down the amount of IPTG, of course). Then lyse the cells ( : before and after induction) and run lysate on a SDS-PAGE gel to check : expression level.
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T**********t 发帖数: 1604 | 5 I meant the amount of IPTG, not the concentration of IPTG... If you use much
less volume of culture (5ml vs. 1 L), the amount of IPTG needs to be scaled
down accordingly.
【在 n***w 的大作中提到】 : Actually many protocols say high concentrations of IPTG can be used at : screening stage, up to 1mM. : : used : I
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s********n 发帖数: 2939 | 6 我都是每一次都重新做transformation,平板4度保存,大概可以用2-4周。
大部分Novagen的protein expression strains都是recA+,质粒不稳定,虽然保存在-
80C,接种多次以后难免会有影响。我知道至少有3个negative的例子了。
如果你坚持要这么做,有一个相对low risk的方法,就是用含有葡萄糖的培养基培养,
分装成许多小管,0.5-1 ml (或更大体积),-80C保存,每次拿一管直接培养做表达
,不要多次冻融。
from
【在 H*g 的大作中提到】 : Sorry only could type in English using lab computers : I a trying to express protein in E.coli and am wondering if I should save : glycerol stocks for transformed : BL21(DE3) or Rosetta2(DE3) strains for future or should we always start from : a new transformant? : How often does mutation occur in these strains? : Thanks a lot!
|
w**t 发帖数: 52 | 7 接种不用反复冻融吧,我都是n从-80拿出来就用tips挑一点冰渣划在板上。动作快点就
好了
【在 s********n 的大作中提到】 : 我都是每一次都重新做transformation,平板4度保存,大概可以用2-4周。 : 大部分Novagen的protein expression strains都是recA+,质粒不稳定,虽然保存在- : 80C,接种多次以后难免会有影响。我知道至少有3个negative的例子了。 : 如果你坚持要这么做,有一个相对low risk的方法,就是用含有葡萄糖的培养基培养, : 分装成许多小管,0.5-1 ml (或更大体积),-80C保存,每次拿一管直接培养做表达 : ,不要多次冻融。 : : from
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s********n 发帖数: 2939 | 8 到是没有溶解,还是凝固的,但温度还是升高了不少。Anyway,我是由于周围有不少
negative的例子,所以不这么做的,并不是说所有都会出问题。
【在 w**t 的大作中提到】 : 接种不用反复冻融吧,我都是n从-80拿出来就用tips挑一点冰渣划在板上。动作快点就 : 好了
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H*g 发帖数: 2333 | 9 For the negative examples that you mentioned, since the transformed E.coli
could survive antibiotic stress, so
the plasmids are still there. So the researchers discovered mutation(s) in
the coding region of protein
interested in those plasmid(s)?
【在 s********n 的大作中提到】 : 到是没有溶解,还是凝固的,但温度还是升高了不少。Anyway,我是由于周围有不少 : negative的例子,所以不这么做的,并不是说所有都会出问题。
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s********n 发帖数: 2939 | 10 我所说的negative不是指质粒丢失了,一个例子是质粒变大了,估计是重组了,蛋白表
达量下降很多;另外两个例子都是蛋白不表达了,估计他们都没有测序,只是重新转化
。 |
y*****1 发帖数: 73 | 11 哈哈!! 1mM means 1mM final concentration in the culture....
much
scaled
【在 T**********t 的大作中提到】 : I meant the amount of IPTG, not the concentration of IPTG... If you use much : less volume of culture (5ml vs. 1 L), the amount of IPTG needs to be scaled : down accordingly.
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