b******y
发帖数: 627 | 1 If you are doing protein expression, you don't need to measure OD600. I
always estimate by eye. That is because I never follow induction at OD600 =
0.6 rule. This rule is bullshit for a lot of, if not most, proteins. So far,
I have worked on more than 100 proteins (and their variants). None of them
require induction at OD600 = 0.6.
Normally I estimate the OD600 at 0.6~0.8. I reduce the temperature to 16~20
degree for an hour. Add 2 gram/liter of glucose and 1 mM IPTG. Don't worry
too much if you... 阅读全帖 |
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d********r
发帖数: 3279 | 2 Arsenic-associated bacteria (NASA's claims)
ResearchBlogging.org
Wolfe-Simon F, Blum JS, Kulp TR, Gordon GW, Hoeft SE, Pett-Ridge J, Stolz JF
, Webb SM, Weber PK, Davies PC, Anbar AD, & Oremland RS (2010). A Bacterium
That Can Grow by Using Arsenic Instead of Phosphorus. Science (New York, N.Y
.) PMID: 21127214
Note to new readers: I wrote this post on Saturday Dec. 4, mainly to
clarify my own thinking. I didn't expect anyone other than a few
researchers to ever read it. Since then I've made ... 阅读全帖 |
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z*********3
发帖数: 335 | 3 OD600 doesn’t give absolute cell concentration
OD600 is cell dependent
Need to independently measure cell concentration so that the two can be
related.
Measure absolute cell concentration by dilution and plating.
Plating measures cfu
Standard curve = plot OD600 vs. cfu |
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s********n
发帖数: 2939 | 4 从current protocol上改的：
1. check OD600；
2. harvest X ul induced or uninduced culture;
3. add Y ul 1x SDS-PAGE loading buffer (Y=OD600*X/10);
4. Vortex for 30s;
5. 100C for 5 min;
6. 15,000x g for 5 min;
7. Load 5-20 ul supernatant to SDS-PAGE gel.
你也可以向楼上所说，把soluble和insoluble的fractions分离后再SDS-PAGE. |
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l**********1
发帖数: 5204 | 5 GST-A.B co-expression pls let IPTG and the cultures were maintained at 4°C
for 48-72 h then do that induction
reference:
PMID 23525091
Production of soluble eukaryotic recombinant proteins in E. coli is favoured
in early log-phase cultures induced at low temperature. (2012).
Springerplus. 2:89.
FINDINGS:
A high yield of active soluble proteins was obtained by combining early-log
phase cultures and low temperatures for protein induction. When IPTG was
added at OD600 = 0.1 and cultures... 阅读全帖 |
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R*s
发帖数: 2041 | 6 1. never heard of measuring bacterial at OD546 and calculating as u
said below. We only do it at OD600.
2. What temperature you grow the cells? Hope it is 37, not 30.
BL21(DE3)Plyss does grow relatively slower than other E.Coli strains.
However, it is not as slow as yours.
3. In fact, 12 hours for your transformed culture to reach OD 0.8 is not
terribly slow. It usually take us 6-9 hours to do so.
4. Last suggestion, make sure your Amp and Chl concentration is correct.
Even if they are correct, |
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b********c
发帖数: 161 | 7 invitrogen pEXP5-NT/TOPO vector 和 BL21(DE3)Star competent cell
细胞长到 OD600=0.6~0.8时加入IPTG (final C=0.5mM)诱导表达, 25C和37C都试过,时
间点为2h, 4h 和6h. 然后提纯蛋白和跑SDS-PAGE：诱导前，诱导后，提纯后.
由于T7的leaky expression,我仍然提纯到了我需要的蛋白,关键问题是蛋白的量在IPTG
诱导前和诱导后没有变化。请问到底是哪个环节出错了呢？ |
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H*g
发帖数: 2333 | 8 Thank you, guys!
I sequenced my expression plasmid and I am sure it is correct, no frame
shift, no mutations. I feel my protein
is not toxic to E.coli. I shaked the E.coli culture at 37C till OD600=0.6,
then add IPTG to 1mM. I also had a
second culture but not add IPTG.
I took 1.5mL every hour for 7 hours from both cultures (with and without IPTG), spin
down, sonicated, and loaded side-
by-side on SDS-PAGE, I couldn't see any band being induced.
I would try lower temperature and lower concentrat... 阅读全帖 |
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e****s
发帖数: 1125 | 9 2个问题，
1）你检测的是Supernatant还是Total lysate?
2)你是依靠考染对比还是用的Western?
如果你上样的是total lysate，然后Western看不到明显的表达，那就不用试其他条件
了。
如果你只是SDS-PAGE后，用考染，很可能有表达，但灵敏度不够，建议WB对比。
frame
OD600=0.6,
without IPTG), spin |
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n***w
发帖数: 2405 | 10 Thank you guys.
I use PGEX2T vector so GST is on the N-terminus.
I normally do 100ml culture size. Here is how I did this:
1. small culture of the bacteria from glycerol stock O/N, about 6-8ml.
2. transfer the small culture to 100ml 2xYTA medium in the next day morning
, shaking at 300rpm, 37degrees.
3. it ususally takes about 1hr 40min - 2 hrs to have an OD600 around 0.75.
4. add IPTG (stock 100mM) 100ul to get a final concentration of 0.1mM.
5. Lower the temp to 22degrees and shake at 300 rmp... 阅读全帖 |
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s******a
发帖数: 472 | 11 我用的是SW102, 估计不同genetic background的strain对streptomycin的敏感性也不
同。 早先报道rpsL-kana筛选的paper他们用1mg/ml；但是genebridge公司
recombineering kit用rpsL-neo，他们用50ug/ml。怎么有这么大的差别呢。
我后来测了一下OD，2.5mg/ml streptomycin只能使OD600降低20%左右。
streptomycin不能完全抑制细菌生长，但是到底多大溶度才合适进行counter-
selection呢？ |
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D**A
发帖数: 311 | 12 用细菌表达蛋白，0d600大约1.54的时候才加iptg，这样会不会蛋白就不表达了呢？
谢谢 |
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l*********1
发帖数: 351 | 14 对于某些非可溶蛋白，可以高OD（1或者2），短时间诱导。ge healthcare有相关建议
的。 |
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