m******5
发帖数: 1383 | 1 Did anyone here used plasmid or cDNA clones from OriGene?
is their product reliable?
Moreover, in many of their plasmid they denote the plasmid are not
replicable by regular DNA purification method, is this possible? Looks to me
they are just trying to get customers to buy the plasmid from them instead
of purifying the plasmid indefinitely in their own bench
I attached their 'explanation below'
;Due to the inherent nature of this plasmid, standard methods to replicate
additional amounts of DNA ... 阅读全帖 |
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w**t
发帖数: 52 | 2 The streaking do not 'dilute' the plasmid, but simply purify the colonies.
The copy number of the plasmid mostly depend on your replication origin (
never use 2u origin if you want a low copy), and whether the plasmid contain
centromere (like CEN6 on pRS416). Usually the centromere sequence can limit
the copy number of your plasmid.
I don't think you need to wash or replica your plates. If you don't want to
have too many plasmids in one cell, just simply reduce the transformation
efficiency and ... 阅读全帖 |
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l**********1
发帖数: 5204 | 3 pVITRO1-neo-mcs
neo (kanamycin/G418 resistance).
pVITRO1-MCS
Expression plasmids pVITRO1-MCS are developed mainly for in vitro studies.
pVITRO1-MCS allow the ubiquitous and constitutive co-expression of two genes
of interest.
pVITRO1-MCS plasmids can be stably transfected in mammalian cells and the
genes of interest are expressed at high levels.
pVITRO1-MCS plasmids carry two elongation factor 1 alpha (EF-1α) promoters,
from rat and mouse origins combined to the CMV and SV40 enhancers
respective... 阅读全帖 |
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M*****n
发帖数: 16729 | 4 I need subclone a big promoter, which makes the plasmid over 30 kb.
What kind of bacterial strain should I use to propogate the plasmid and for
subcloning?
What kind of plasmid preparation kit should I use for the plasmid?
Thanks a lot for sharing your experience. |
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M******k
发帖数: 84 | 5 When we say “copy number” of a plasmid, is it the average number of
plasmids per
cell or the maximum number of plasmids per cell?
It possible to lower the copy number of a certain plasmid by using less DNA
during
transformation?
Thanks! |
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y******y
发帖数: 107 | 6 一个plasmid (A) 里面混有gapped plasmid (B)，A和B的序列完全一样，A 和 B的唯一
不同是B一条链上有一个 50bp的gap，A和B都是100 ng左右，希望比较干净的除去B （
只要切断单链区域就行了），然后做PCR扩增A （扩增区域含有B的gapped
region）。目的是希望PCR模板是从A来的，而不是B来的，谢谢。
酶切可以吗？什么酶可以专一切断gapped plasmid中的单链区域？ |
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W**S
发帖数: 275 | 7 今天悲摧了，generate了一批LENTIVIRUS，结果PURO SELECT之后CELL都死了，这次着
急，没做MIDI PREP，直接用MINIPREP的PLASMID做的TRANSFECTION, PLASMID的质量不
是太好，浓度很低，260/230不太好，同一批用MIDIPREP GENERATE的VIRUS没问题。请
问LENTIVIRUS不WORK会是因为PLASMID的质量不好吗？以前也用MINIPREP的PLASMID做过
，没问题。多谢！ |
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m*********D
发帖数: 1727 | 8 看你的selection marker和要表达的基因是不是在同一个vector上。不是的话，你两个
都要切。
以前作knockout的时候，读过的资料是这么解释的：所有integration都是因同源
sequence导致的。一个圆的plasmid,一个点和genoome的一个点match后，integration
往往就是一个copy进去，而一个线性的DNA片断，往往会有很多拷贝进同一个点。你要
有两个plasmid，你就只能切了，不然，两个不同点的integration很难同时在一个细胞
里发生。我作stable,都切，希望一下子能多进几个拷贝。另外，环状的plasmid的
integration可以发生在plasmid的任何地方，如果正好发生在你的expression
cassette之类，就是integrated,也可能不表达你要的东西。
切点很容易，就是找一个酶位点，只要在你的expression cassette（包括你自己的基
因和selection marker)之外就行。你知道vector的来源的话，一般公司的网站上会有
map告诉你这些cassette的起点和终点的... 阅读全帖 |
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w**t
发帖数: 52 | 9 All depend on your transformation efficiency. If you are talking about co-
transforming different plasmids, I will say it is not easy to co-transform 4
plasmids at the same time. And if you are doing library transformation,
there will be many different plasmids in one cell. The traditional way to purify
your transformants is streaking the single colony for 2-3 times. |
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m******5
发帖数: 1383 | 10 expression plasmid linealized,不带任何selection gene
遂决定共转其与一个selection plasmid制作stable transfect cell line
求protocol
最好电转可用的 |
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d****i
发帖数: 2346 | 11 今天用Attractene转染plasmid，先加plasmid到optim medium，然后加Attractene，然
后去vortex和spin down。发现管底部有白色沉淀。用手指弹了弹管子，白色沉淀慢慢
消失了。
plasmid浓度为1ug/uL. A260/280=1.9, A260/230=2.3. 溶解在10mM Tris-Cl里面，离
心无沉淀。
Attractene是2015年1月买的。单独加Attractene到optim里面离心无沉淀。
那么那个白色沉淀到底是什么？ |
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B****n
发帖数: 22 | 12 I tried to prepare some plasmid dna for an episomal plasmid(20kb), but didn'
t get anything with normal maxiprep kit from MACHEREY-NAGEL.
Anyone has some experience with this kind of large plasmid dna preparation? |
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m******5
发帖数: 1383 | 13 大家出现过大plasmid每一批提纯跑出来的条带大小都不一样，更甚而每一次跑胶大小
都不一样的情况么？我正在做大plasmid连接，十分困惑 |
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m******5
发帖数: 1383 | 14 I am doing a large fragment(2.3) plus large vector(12k) ligation.
I have learned previously that some plasmids containing certain sequence may
not be stable in DH5a line, but I didn't find online about which kind of
sequence or plasmid would be unstable.
Does anyone here have experience on handling this problem? |
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e**r
发帖数: 1144 | 15 查了一下手头的transfer plasmid的map,似乎是没有
packaging plasmid没有图
如果加一个SV40 ori, 会不会能提高titer ?
应该有人试过吧? 谁看过相关的文献? |
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X***n
发帖数: 366 | 17 Single strand DNA plus small amount of plasmid, do red recombineering.
Google.
请问高人，有没有什么办法在一个22kb的plasmid中特定位置插入30 bp的序列？谢谢
★ Sent from iPhone App: iReader Mitbbs 7.28 - iPad Lite |
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e***o
发帖数: 344 | 18 各位做酵母转化时一般一个细胞里能转进多少个不同的plasmid，等酵母涨到比较大的
克隆时估计还有多少种plasmid
不知有人是否注意到这个没有，谢谢 |
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y*********u
发帖数: 183 | 19 请教大拿一个问题
手上有一个30k大的BAC transgenic plasmid
构建时想过可以在culturing cell上试验，于是引入了一个SV40 replication Ori
目前刚拿到plasmid，准备表达，请教一下用哪个转染试剂比较好？
听说Lipofectamin LTX行？ |
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g*********5
发帖数: 2533 | 20 do someone use a mammalian plasmids that have two promoters and could
express two SEPARATED RNAs? I need express one protein and one mRNA in the
cells with one plasmids. thanks. |
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d**p
发帖数: 149 | 21 Why do not modify your current plasmid? I did a lot modification by myself
to meet the clone requirement. There is no perfect plasmid for every purpose.
有 |
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s******y
发帖数: 28562 | 22 在我的理解中，动物身体上的细胞有一定的能力来随机吸收plasmid,只是效率不高而已
plasmid |
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h*********g
发帖数: 18 | 23 我用miniprep提取plasmid，得到的DNA太少，后悔没用maxiprep，(I cannot find the
original plasmid) 请问有什么办法补救吗？谢谢。 |
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h*********g
发帖数: 18 | 24 I got several plasmids from other lab and need to clone them and then do
maxiprep.
We have TOP10F' E.coli.
I will add 2 ul plasmid (100ng/ul) into one tube of E.coli TOP10F', mix, put
one ice for 5-30 min. Then heat-shock for 1 min at 42 degree.
Transfer the tube on ice. Add 250 ul room temperature SOC medium. shake the
tube at 37 for 1 hour.
Then spread 10-50ul onto a plate.
Is that right? I am new to cloning. Please help. Thanks. |
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h*********g
发帖数: 18 | 25 我用maxiprep提取plasmid (2Kb)，跑胶得到2条带，还有些模糊的带，cut后也有几条
带，其中一条带是2kb, all the other bands are larger than the 2kb band。THe
restriction enzyme should only cut one site in the plasmid.Can I trust the
result? After that I will do the gel extraction. Why there are several bands
? Thanks. |
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f*****r
发帖数: 84 | 26 是一个library 的PLASMIDS, 所以跑胶会像smear，切一大块胶不知道产率会不会很低
呢。
exonuclease对plasmid 没影响么？
thanks! |
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f*******e
发帖数: 628 | 27 很多 plasmid extraction kit 里面在初期提纯的步骤都会用 exonuclease 来去除
gDNA。
因为 plasmid 是 circular 的，不会被影响。之后的进一步提纯步骤会同时去除掉
exonuclease. |
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C***Y
发帖数: 142 | 28 如果哪位大侠有sigma-1 receptor plasmid, 敬请站内PM我。最近需要overexpress
sigma-1 receptor. 万分感谢！！！ |
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C***Y
发帖数: 142 | 29 如果哪位大侠有sigma-1 receptor plasmid, 敬请站内PM我。最近需要overexpress
sigma-1 receptor. 万分感谢！！！ |
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w**u
发帖数: 311 | 30 各位XDJM，借人气问一问题
我一朋友半年前海归，留了两盒`质粒让我保存`。现来电让我用他的FEDEX帐号寄回去
。不知道出入海关会有限制和麻烦吗？各位有无经验如何填FEDEX的“shipment
information"栏
我如下填写：
Commodity Description：Plasmids in dry ice
Harmonied Code: 不知怎么写
Country of Manufacture: USA
Value of Customs: N/A
自己很想帮忙，但也不想给他或我带来麻烦。
请前辈们指教 |
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e**********m
发帖数: 397 | 31 正解。
没听说过用dry ice邮寄plasmid的 |
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O*C
发帖数: 649 | 32 刚发3个月的新文章，没有什么citation，只有不少人找老板要plasmid的email，老板
forward给我。这种能算好的contribution的证据么？ |
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d*******n
发帖数: 4778 | 33 应该算呀！
你构建的plasmid,可以描述一下它的用途，当然最好是表达载体，你怎么设计的，怎么
retieved你感兴趣的gene的等等。然后很多相关领域的scientists如何如何...
^_^ 很多东西，看你怎么说了。 |
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c*********e
发帖数: 1 | 34 1. Never expect to isolate a large amount Plasmid DNA from yeast.
2. What I did is:
Incubate lytic enzyme with your yeast for an hour. Then followed the
BioRad kit miniprep protocol, start from lysis solution (equal to solutionII
in classical protocol). I guess kits from other companies are also work.
At the end, elute your DNA from matrix with 20microliter of TE instead of 100
microliter. One microliter of such extract is sufficient for a bacteria
transformation, even though it's CEN plasmi |
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p*****n
发帖数: 981 | 35 ☆─────────────────────────────────────☆
iamabigpig (温暖企鹅) 于 (Fri Feb 2 16:49:49 2007) 提到:
Just constructed two new revised plasmids and found that the copy number for
one is really low in the cells. Any method to increase the copy number?
change the ori? Thanks!
☆─────────────────────────────────────☆
HENBEN (benben) 于 (Fri Feb 2 17:11:12 2007) 提到:
Use chloramphenicol. Check Molecular Cloning for details.
☆─────────────────────────────────────☆
iamabigpig (温暖企鹅) 于 (Fri Feb 2 17: |
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s******y
发帖数: 28562 | 36 I am preparing a low yield plasmid and I need a lot of it.
I want to use the chrolamphenicol but I forgot the protocol because I have
not done it for a long, long time.
I roughly remember I should add the chrolamphenicol to the overnight culture
and then continue for some time, but I forgot what concentration of
chrolamphenicol I should use and how long I should incubate the bacteria
with the chrolamphenicol.
Any info would be appreciated. Thanks in advance! |
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a****d
发帖数: 1919 | 37 maybe they are talking about packaging plasmids mix, or something like that.
But still, you can figure it out. But if you have to use specific enzymes
for the reaction, like the pENTR system from invitrogen, then you have to
pay for the reagents. |
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s*******l
发帖数: 2445 | 38 谢谢两位的回复， 再请教一下packaging plasmids mix大概是怎么回事？ |
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y******y
发帖数: 107 | 39 手头上有一个 pBABE-puro SV40 LT plasmid ，是从addgene买的， 图谱如下连接
http://www.addgene.org/13970
请问一下这个plamid能否表达 SV40 large T antigen( LT). 因为从图谱上看是 SV40
LT gene是插在BamH I位点之间，可是这个SV40 LT gene是在SV40 promoter 之前，另
外一个比较奇怪的问题是为什么有两个SV40 promoter，多谢！ |
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s********n
发帖数: 2939 | 40 滤纸片很方便，但是不能放太长时间，之前别人给了我十几个plasmids的滤纸片，在-
20C放了一个月，结果有1/3完全不能recover. |
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m*****a
发帖数: 26 | 42 Just use small PCR tubes (with a lot plasmids) and hide in a book(dig the
book to leave a small hole inside).My boss used this way. |
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o**i
发帖数: 1165 | 43 plasmid addgene挺好
cell coriell也可 |
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M******t
发帖数: 555 | 44 恩,准备从BAC中选一段大约15KB的region克隆到retrieval vector.但关键是还得做个
point mutation.这么大的plasmid很没把握,有什么好的技术推荐吗?谢谢. |
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C*******e
发帖数: 4348 | 45 Invitrogen 的DH10B是适合做大Plasmid还有cosmid的
不好的是本身有Sp抗性
for |
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X***n
发帖数: 366 | 47 1. Average number. There is little difference between average and maximum.
2. No. It depends on the replication origin of the plasmid. |
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v***a
发帖数: 1242 | 48 我在研究某个蛋白在其overexpress情况下对某个器官的作用。想给老鼠注射含有这个
蛋白insert的plasmid DNA，第一次做，不知道有些什么需要注意的？是不是可以用PBS
作为等价于in vitro中溶解DNA的buffer/media？我在in vitro中overexpress的时候，
用的transfection reagent是QIAGEN的effectene，不知道给老鼠注射的话也仍旧可以
用这个吗？或者大家有推荐的吗？
非常感谢！ |
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n*****s
发帖数: 8 | 49 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 |
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s******r
发帖数: 2876 | 50 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 |
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