x*****o
发帖数: 441 | 1 提纯细胞,IPTG induce 之后5小时生长,之后就是离心.我的问题是离心之后大家都怎
么resuspend 细胞. 我同事说用磁子,搅拌.不过她说这个她自己发明的,别人都是冷室
里面用移液管上下吸溶液resuspend 细胞.她嫌太慢,就用磁子,快.
用磁子和用移液管,哪个好呢?
谢谢! |
|
g*********5
发帖数: 2533 | 2 Isolation of nuclei for the co-IP and whole ChIP protocols is based on the
methods of Gendrel et al. (2002, 2005), Johnson et al. (2002), and Nelson et
al. (2006).
METHOD
For extraction and co-IP of nuclear proteins, see Steps 1-39 (Fig. 1). For
the ChIP procedure, see Steps 40-69 (Fig. 2).
Figure 1.
View larger version:
In this page
In a new window
Figure 1.
Flowchart for the timeline and organization for co-IP of nuclear proteins
from Arabidopsis seedlings.
Figure 2.
View larger versio... 阅读全帖 |
|
h**********r
发帖数: 671 | 3 我在实验室用的Thermo，貌似work的不是很好。
Yeast DNA Extraction
(The protocol was modified by housepainter on December 28, 2009 according to
manufacture’s instructions)
Yeast DNA Extraction Kit, Thermo Number Description: 78870
Storage: Store at 4°C; if precipitant has formed, gently warm DNA Releasing
Reagents A and B at 37°C for 1-5 minutes.
Kit Contents:
Y-PER Reagent, 25 ml
DNA Releasing Reagent A, 20 ml
DNA Releasing Reagent B, 20 ml
Protein Removal Reagent, 10 ml
1. Pellet a 10 ml S. cerevisiae culture gr... 阅读全帖 |
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g*****y
发帖数: 6325 | 4 我以前做的membrane extract 的protocol 超级简单。 1. 用pbs resuspend 细胞，
sonicate; 2. 2000rpm 2min 取supernatant. 3. ultra centrifuge 28000 rpm 1hr;
4. 除去上清，用你的lysis buffer resuspend pellet. 然后加 loading buffer 跑胶
。 |
|
b******k
发帖数: 1874 | 5 I am writing to ask for help.
I am using Miltenyi anti-NKp46-biotin-bead- MS column kit. The first step is
to get splenocyte from spleen by gentlMACS without enzyme treatment. A very
simple protocol, just put spleen into the PEB buffer and let gentleMACS run
m_spleen_01 program once, and filter, spin, aspirate, resuspend the pellet.
The problem comes from the resuspending process. The small tight pellet will
produce(or contain) big clump that is almost impossible to break when 10 ml
PEB buffer i... 阅读全帖 |
|
h**********r
发帖数: 671 | 6 我以前在国内时候提细菌基因组用的方法，貌似对酵母也work,我以前就提过一次酵母
，确实work的很好，还记得gel上的照片贼亮贼亮的。那时候年轻啊！问过别人，说
lysozyme对yeast也能work.
Extraction of DNA from Bacteria and Yeasts
(This protocol was developed by Housepainter on February 09, 2010)
1.Inoculate 5 ml of medium with a single colony of transformed bacteria or
yeasts.
2.Pour 1.0 ml of the culture into a tube. Centrifuge at maximum speed for 30
seconds at room temperature. Remove the supernatant.
3.Resuspend the bacterial pellet in 1.0 ml 0.85% NaCl by vigorous vortexi... 阅读全帖 |
|
s******y
发帖数: 28562 | 7 Polysome purification and fractionation
(Based on Davies & Abe, 1995, Methods in Cell Biology, Vol 50, Chapter 15)
1. Prepare 4.6ml 15-60% sucrose gradients in 5ml polyallomer tubes (Beckman
#326819) by
pipetting 2.3ml of 60% sucrose in buffer B followed by 2.3ml of 15% sucrose
in buffer B
(slowly, drop by drop, against the wall). Cap the tubes with rubber stoppers
and lay tubes
horizontally for 3h @RT for sucrose to diffuse (or for 2h if making 10-40%
gradient). Place the
tubes again vertically... 阅读全帖 |
|
g*******m
发帖数: 42 | 8 A) Ethanol precipitation
1)Add to the DNA solution 1/10 volume of 3M NaOAc and 3 volume of EtOH
2)Leave in the -70 degree freezer for 20 minutes
3)Spin in microcentrifuger for 10 minutes to pellete DNA
4)Wash with 70%ethanol and spin for 10 minutes
5)Air-dry the DNA and resuspend in water,Tris buffer or TE buffer
B) Isopropanol precipition
1)Add to the DNA solution 1/2 vol.of 7.5 M NH4OAc and 2 vol. isopropanol
2)Incubate at room temperature for 10 minutes
3)Spin for 10 minutes.Wash with 80%et |
|
l*****g
发帖数: 263 | 9 use needle pass-through bah
Use low ionic strength buffer (Tris 20mM pH 7.7, protease inhibitor mix, MeSH)
(i.e. Buffer) and 27.5G needle (i.e. Needle)
1. wash cell with PBS, spin down cell, remove all PBS.
2. resuspend cell in Buffer, pipete 10 times, sit on ice for 15 min.
3. use shringe and Needle, pass the suspended cell through Needle 7 times
4. site on ice for another 10 min.
5. pass through Needle another 7 times
6. spin at 500 g to remove debris, 100k g for membrane fraction |
|
s***l
发帖数: 42 | 10 1.start from 3-4ml fresh overnight culture, pellet cell in 8000rpm in cold for
3min
2.resuspend in 200ul P1
3.lysis with with 400ul P2 <5min in RT
4.neutralize with 300ul P3 on ice for 5 min
5.spin down 10min at maxi spped in cold
6.supernatant to a new tube
7.add 0.75ml cold isopropanol
8.spin at maxi speed RT for 15min
9.discard supernatant, wash with 0.5ml ethanol, spin 5min maxi speed in cold
10. asirate supernatant and air dry DNA till pellet starts to turn translucent
Caution: DON'T OVER D |
|
s******y
发帖数: 28562 | 11 我用的是embryonic fibroblast, 和3T3差不多。
一般都能做到20%。 再高就没有办法了。
基本上，你要确认两件事：
1。你提的DNA足够纯。如果不放心的话，可以用ethanol/NaAc 沉淀法
纯化一次
2。细胞的状态。 必须在60~80% confluence 之间。
如果都不行的话，还有一个不是办法的办法，
那就是把细胞trpsinized, wash, resuspend, 然后在细胞贴壁之前
把转染混合物加进去。因为细胞在spread and adhere 的时候会吸进很多
vesicle.相当于就是强行把DNA弄进去了。不过这个方法会弄死很多细胞。
谢。 |
|
T*****u
发帖数: 3257 | 12 if you just want to pass the cell, you can just take some cells with the old
medium and add them to the new medium.
if you want to completely change the medium, y ou can centrifuge the cells
down and resuspend them in new medium. |
|
s******y
发帖数: 28562 | 13 Easy. Put the cell in 50mL tubes.
Spin the cell down. Resuspend in new media
be
change |
|
s******y
发帖数: 28562 | 14 yes. For example, a recent paoer in Nat Med, people in Harvard has made a
functiomnal lung that last for a few hours. Of course, it is not from iPS or
other stem cells. It was made from a washed out matrix and a bunch of
resuspended lung cells.
http://www.reuters.com/article/idUSTRE66D0C220100714 |
|
e****s
发帖数: 1125 | 15 90% methanol precipitate
wash one more time,
then resuspend by sample buffer.
loading |
|
j*********o
发帖数: 237 | 16 After linearization, extract the DNA as follows:
-1X Phenol
-1X Phenol:Chloroform
-2X Chloroform
-Precipitate with EtOH and resuspend in water at a final concentration of
about 1g/l |
|
n*****s
发帖数: 8 | 17 I used Qiagen plasmid plus Maxiprep kit to purify my construct. And I was
asked to do a second precipitation after the elution step in order to
further remove the salt contamination. I use -20C isoproponal centrifuge at
room temp for 30 min, 15000xg, then wash with 70% ethanol, and resuspend
the DNA in EB buffer.
I did not see the precipitation at the bottom of the 2ml tube after the 30
min, a senior phd student told me that sometimes even one can not see the
precipitation one will still get so... 阅读全帖 |
|
s******r
发帖数: 2876 | 18 The bacteria may be over grow,
try to pick up a new colony.
I used Qiagen plasmid plus Maxiprep kit to purify my construct. And I was
asked to do a second precipitation after the elution step in order to
further remove the salt contamination. I use -20C isoproponal centrifuge at
room temp for 30 min, 15000xg, then wash with 70% ethanol, and resuspend
the DNA in EB buffer.
I did not see the precipitation at the bottom of the 2ml tube after the 30
min, a senior phd student told me that sometimes... 阅读全帖 |
|
S*******m
发帖数: 34 | 19 You can save some time by adding plasmid mixture (immediately after
preparation) to trypsinized and resuspended 293T cells. Worked pretty
well for me.
To save some money, you can also prepare a batch of HBS with different
pH and test which one works best for you.
confluency
reach 40
fresh |
|
|
|
n***w
发帖数: 2405 | 22 Hi, all,
I am using Rosetta-Gami DE3 pLySs to expression my fusion protein which is
predicted as 91kDa.
I first did the expression assay and found protein expression was induced
after adding IPTG by testing the whole cell lysate. (bacteria at certain
time points, add 2X sample buffer, boil, SDS-PAGE).
Then I moved to culture more and lysed the cells using a sonicator, after
resuspending the pellet with 1x cold PBS/1% PI. After all these, I loaded
the supernatant and pellet side by side and stain... 阅读全帖 |
|
s********n
发帖数: 2939 | 23 Then I moved to culture more and lysed the cells using a sonicator, after
resuspending the pellet with 1x cold PBS/1% PI. After all these, I loaded
the supernatant and pellet side by side and stained by GelCode and a bulk of
protein (I assume it was protein) was detected in the pellet part.
你有没有在supernatant中检测到你的target protein？如何检测的？WB or activity?
从你的表述你好像没有做WB。
Then I tried 1L culture and repeated the 2nd experiment. This time, I
incubated the supernatant with glutathione sepharose 4b beads an... 阅读全帖 |
|
n***w
发帖数: 2405 | 24 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 |
|
h**********r
发帖数: 671 | 25 同上另外一种方法。
Yeast “smash-and-grab” DNA prep:
• pipette 1-2 ml YPD onto transformation plate; scrape colonies off
with the end of a glass slide and pipette into a microfuge tube
• spin down 15 sec; pipette off sup; if cell pellet is more than 50-75
μl, remove excess and discard
• to cell pellet add 0.2 ml lysis buffer, 0.2 ml phenol/CHCl3 and 0.3
g 0.45-0.5 mm glass beads* (I use calibrated scoop made from cut microfuge
tube pierced with syringe needle) – seal carefully because be... 阅读全帖 |
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b*********b
发帖数: 64 | 26 I use QIAGEN Plasmid Midi kit. 100ml culture could give about 30-50ug. I
tried Qiagen's large construct kit.But in my hand, the midi kit is much much
better than the large construct kit, and also takes much less time.The
following is the protocol I got from Qiagen.
User-Developed Protocol:
Isolation of BAC DNA using the QIAGEN® Plasmid Midi Kit
This procedure has been adapted by customers from the QIAGEN® Plasmid
Midi Kit Protocol.It has not been thoroughly tested and optimized by QIAG... 阅读全帖 |
|
c********b
发帖数: 363 | 27 每个步骤都是别人都知道的,我只是组装了一下. 最大不同是别人是先Urea变性,提出来
再复性.我是利用Triton X-100可以洗脱SDS的特点利用SDS变性,然后Triton X100洗脱
复性,然后过柱. 肯定不会对所有蛋白适用,但是起码我遇到的都work (包括一个>100
Kd的酶).
我是用的sonication water bath, 也可以用probe sonication再加SDS,都OK,但是注意
dilution的比例和体积.
Good luck. 用的高兴分我几个包子.
Key, keep a fraction from every step for analyses and trouble shooting
- Culture the E. coli overnight
- Induce it at a proper concentration of IPTG, room temperature for ~
4hrs
- Pellet the E. coli at 6000 rpm at 4 degree
- R... 阅读全帖 |
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F******S
发帖数: 13 | 28 请问你用来resuspend pellet的native lysis buffer是怎么配的， QIAGEN的PROTOCOL
里面有0.3M NaCl， 你再加1% SDS 岂不是全都是沉淀。
assay, |
|
c********b
发帖数: 363 | 29 我是先resuspend之后再加的SDS，冰上放着没有看到沉淀。
PROTOCOL |
|
E**********1
发帖数: 73 | 30 Hi all:
Recently I planning to do siRNA, but I have a detailed question.
I got siRNA from Dharmacon, and it came as dried pellet, which means I need
to dissolve it in some solutions. The instruction suggests resuspending the
dried pellet in RNase-free 1X siRNA buffer (60mM KCl, 6mM HEPES-KOH, pH7.5,
0.2mM MgCl2), or in RNase-free water for short-term storage.
It will take time to place another order only for those buffers. So I am
wondering how you guys do that. Can we make the buffer by ourselv... 阅读全帖 |
|
h*****G
发帖数: 113 | 31 hello,
最近需要用cell surface marker 细胞染色，sort positive 的细胞，然后重新plate
培养。每次sort完细胞，重新plate以后，细胞总是大量死亡。希望大家有什么建议。
下面是我的protocol
Trypsin cells. Stop reaction. collect cells. Wash once with PBS+0.2% BSA.
Staining cells in the dark at RT for 25 min. Staining buffer: PBS (w/o Ca2+
Mg2+) + 0.2% BSA+ 0.09% NaN3.
Wash three twice. (500gX5 min every time)
Resuspend cells in Staining buffer. Sort cells..
Replate positive cells. |
|
m*****z
发帖数: 1451 | 32 concentrate virus and resuspend in PBS |
|
s******a
发帖数: 472 | 33 Why my DNA resuspended in TE looks sticky? |
|
s****n
发帖数: 234 | 34 Microwave method
(Use this for checking integrants)
Smear fresh colony using pipette tip on bottom/walls of a small PCR tube.
Microwave PCR tubes or strips (without lids) for 90 sec in PCR rack.
Resuspend dried out yeast in 25 ul of PCR reaction mix (with Taq already
added) and perform PCR.
Note: Do not overload PCR rack when microwaving--no more than 3 strips
of 8 tubes per microwaving or heat dispersal becomes an issue.
Alkaline lysis method
(Use this for cloning or for checkin... 阅读全帖 |
|
i***l
发帖数: 1656 | 35 determine pH of your 1 liter LB
spin down 10ml ecoli, resuspend with fresh media before transfer
lack of O2? there is special flask for large scale biomass culture
colony contaminated and outgrown in the 10ml tube?
growing |
|
L****S
发帖数: 76 | 36 three possible ways to solve this problem:
(1) Coat the 12-well-plates with vitrogen before plating cells:1/45 dilution
in 1X PBS overnight; 420μl/well;
vitrogen is from Advanced BioMatrix, Inc (5005-B)
Aspirate the nitrogen/PBS before plating cells
This will increase the adhesion.
(2) Collect used medium together with cells when you change medium, then
centrifuge at 1000 rpm (about 500 g, I am not sure, but any speed can pellet
cells without breaking should be ok) for 5 minutes, vacuum the medi... 阅读全帖 |
|
a********k
发帖数: 2273 | 37 这个的全文也就是一个abstract。
OPTIMIZATION OF RECOMBINANT STREPTOLYSIN-O EXPRESSION
AND ANALYSIS OF FINAL PRODUCT FORMULATIONS SUITABLE FOR
IMMUNODIAGNOSIS.
B Velázquez, H Massaldi and A Chabalgoity. Department of Biotechnological
Development, Institute of Hygiene, Av. Alfredo Navarro 3051, Montevideo,
Uruguay.
E-mail: b***[email protected]
The streptolysin-O gene of Streptococcus pyogenes was cloned in pGEX-2T and
expressed in Escherichia coli, with the aim of using the product in
immunoassays.
Expression... 阅读全帖 |
|
C*******e
发帖数: 4348 | 38 最近做一个蛋白A
高pI（>9)
已知蛋白A跟蛋白B，或者蛋白B+C有相互作用
于是共表达然后纯化复合物
纯化过程中用detergent啊，lyzosome啊
DNase还有RNase啊
结果破碎细胞以后离心得到的pellet，还有得到的复合物pellet都超级难resuspend的
一整块像Jelly的东西
能弄成大小碎块但是没有办法混匀
用针头反复吸吹也不行
加Uear到5M也不行
参考了一些提纯histone protein的protocol，
加HCl到0.25M也没有办法完全溶解
求建议！ |
|
b*****s
发帖数: 169 | 39 做酶切鉴定或是测序，我从来不用kit，效果是good enough，已经测序有近2000个样品
了。只有做转染才用kit。
我的方法是挑菌落到1ml LB in 1.5ml eppendorf tube, culture for 16h, 吸取50-
100ul culture in 96-well plate 放在4oC 用于以后正确克隆的扩大培养。离心
13200rpm for
0.5min. The pellet is resuspended in 200ul P1, then P2, then P3, spin at
13200rpm for 15min after stored at 4oC for 30min. add equal volume
isopropanol to the supernate in a new tube, spin for 15min, wash with 70%
ETOH. 然后溶于50-100ul水或是TE即可，浓度在150ng/ul 左右。一天做96个克隆是不
难的。
这种样品测序时我试过两个公司，同一样品，genewiz可以得到23个有用的... 阅读全帖 |
|
v**********t
发帖数: 9 | 40 从Dharmacon订的RNA，25mer, single-strand, HPLC purified, deprotected,
desalted.
拿到手后用DEPC water resuspend, 加到蛋白溶液后就把蛋白给沉淀了。先后订的三
批RNA全这样。
哪位知道这是怎么回事？ |
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n***w
发帖数: 2405 | 41 check this article for methods.
http://www.ncbi.nlm.nih.gov/pubmed/8621645
basically, if you have pellets from 10cm plates, resuspend the pellets in
500ul 500 mM sodium carbonate (pH 11), sonicate (3X/15 s). The lysate is
mixed with an equal volume of MES-buffered saline containing 90% sucrose and
centrifuged on discontinuous sucrose gradient (45%, 35% and 5%) for 16 h at
175,000 g and fractions will be collected for analysis.
, |
|
k*****n
发帖数: 323 | 42 try washing your Dynabeads with PBS 0.5% BSA 3 times, then incubate
antibodies in PBS-BSA for two hour. After IP, make sure resuspend beads
totally during each washing step. I never using sperm DNA. I would suggest
you using Pol II monoantibody 4H8 as positive control. Several steps are
critical for ChIP, 1, crosslinking, always using fresh FA, like 16% FA from
Thermo. for cells 15 min crosslinking with rotation would be enough, quench
with 0.125 M glycine for 1 min. 2 sonication, try Branson so... 阅读全帖 |
|
R****n
发帖数: 708 | 43 http://www.pnas.org/content/108/8/3270.figures-only
This is a biochem paper. The supplementary material mentioned how to convert
a Glutamate Acid to 2-Hydroxygluataric acid. But no detailed infor
Quote from the paper
(R)-2-hydroxyglutarate Synthesis. (R)-2-hydroxyglutarate (2HG) was
synthesized by treatment of D-glutamate (Sigma-Aldrich) with nitrous
acid to forma lactone, which was then hydrolyzed with NaOH
solution to form 2-D-hydroxylglutarate. Purity was 93%. Powder
was resuspended in PBS an... 阅读全帖 |
|
v******0
发帖数: 265 | 44 我最近在使用HILIC Column，是bare silica的那种。因为在测试摸条件阶段，所以就
拿一些tryptic digested peptide做sample,但我分出的峰基本上都很难看，很宽，而
且拖尾严重，重复性也不好，有时同一个sample,两天后用同一个column,本来peak
shape还凑或的峰变得又不好了。
我用的是HILIC-MS,mobile phase也试过加ammonium formate, pH基本在3-4，gradient
也试了不少，sample浓度从100fmol injection到10pmol injection都试过，sample一
般都resuspend在90% ACN中 （先用水溶解，再稀释到90% ACN),可结果大多不理想。不
过同样的system, 换成C18 column就非常好。
我在网上看到的一些example似乎都还不错，但大多数用的是UV detector,也有mass
spec的，不过column基本都是Amide 80或者zic-HILIC之类的，好像是有modified,不是
bared silica.
目前手头... 阅读全帖 |
|
v******0
发帖数: 265 | 45 I read many materials, they all say you have to resuspend the sample in high
organic solvent, so most of them use 90%, but I did try one of my peptide
mixture sample in 75%, the peak shape is not improved either. |
|
