r******y
发帖数: 21907 | 1 我的ligation死活不出东西，原来一做就出，现在折腾多次未果，昨天colony PCR除了
带结果质粒酶切后nothing，一定要在节前做出来，生物同修们，有啥建议？我订了新的
ligase，估计是buffer冻容多次就不好了？ |
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r****g
发帖数: 511 | 2 ==b was checking out some other info. and saw those two posts...==b fyi...maybe Tubal ligation for guys after babies' arrival? @@~`
am out of these posts...FYI only... ==b
www.39.net 2008-11-27 39健康网社区
警！常用安全套易患卵巢癌
卵巢癌的死亡率在所有妇科肿瘤中最高，超过宫颈癌和子宫内膜癌总和。天津肿瘤
医院妇科肿瘤科专家提醒警惕卵巢癌变时，将经常使用安全套也列入增加患癌风险。
日本和德国科学家发现，男性精液中含有一种名叫精液胞浆素的成分，不仅可杀灭
女性阴道中很多致病菌，减少生殖道感染几率，还可降低卵巢癌变的风险，如果经常使
用安全套，女性会失去这样的保护机会。
此外，40~60岁的女性如果持续腹胀超过半个月，也可能是卵巢出了问题。深居盆
腔内部的卵巢，一旦长有肿瘤，就可能刺激牵拉盆底韧带，导致腹水、腹胀、疼痛等。
不生育的女性、压力大的都市白领也都是卵巢癌的高危人群。而在直系亲属中， |
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s***t
发帖数: 13743 | 3 你的兴趣很广泛啊。
maybe Tubal ligation for guys after babies' arrival? @@~` |
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S*******w
发帖数: 24236 | 4 思春了？
maybe Tubal ligation for guys after babies' arrival? @@~` |
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n*******e
发帖数: 27 | 5 use NEBuffer2+BSA, digest enough amount of PCR product O/N. i do not know
if you add some nucleotides for buffer purpose. if it is for mutagenesis,
or expression of certain gene, maybe difficult. however, check with BioLab
for Hind3, even 12bp sequence (6bp as buffer flanking) could not be completely
digested O/N. for Xho, it is better, about 75%. so the 1st thing you should
do is to check your design, order new primers if necessary. they are so
cheap nowadays. actually, since it is only 150bp. |
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l*****k
发帖数: 587 | 6 too long to read :)
I think it is possible pet20 have leaky expression, it may kill the cells.
in this case, I think novagen have a cell line that won't have leaky expression.
Instead of cloning disgested fragment, how about do blunt cloning using
novagen's perfect cloning kit? then cut it, at least you won't worry about the
short seq at the restriction site, after cloning,
with multiple clone sites on both side, you
have more choice to put it to pet20, though you have to worry about the problem |
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g***m
发帖数: 465 | 7 my experiences:
this year I tried cloning 4 genes, now only succeeded in 3.
The bad thing is I chose XhoI as one enzyme site for all
those genes, which turned out to be a big mistake. For some
reason, according to an experienced researcher in our lab,
XhoI itself isn't a good enzyme, star activity would be
found often.
For the 3 genes I made progress on, I screened about 200
clones for each construct, the positive rate is
extraordinarily low. 2-3/200 colonies.
Now I am still working on the 4th g |
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r***a
发帖数: 14 | 8 Then count me as the fifth. :)
Your problem reminds me one problem I had before. I kind
of did the similar things, PCR a short piece, about 140 bp.
After I subcloned it into TA-vector and
the sequencing, I found the gene was in the vector except
the restriction sites. Weird. Currently my labmate is having
the same problem, the restriction sites on the ends of
primers are not there although all other sequences seem
fine. Her piece is about 120 bp. Is it related with the PCR
of small pieces? B |
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a***o
发帖数: 129 | 9 not really, you have actually same # of steps as 2-step PCR
One step: PCR----DpnI-----Self ligation
two step: PCR----PCR---TA cloning
And actually your steps are more tricky, if you consider that Pfu has low
processivity while you have to PCR the full length of the plasmid which is at
least 3kb, you didn't really save time; and also compared to the efficiency
of 2-step which is 100%, the efficiency of your method is low; another
consideration may not be so important to you. you have to buy ex |
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M****e
发帖数: 70 | 10 with my experience, the suggestion is to check the PCR product
first. i would rather not digest the PCR product, actually, it
is better if you can subclone it first. for double digest with
NheI and XhoI, if you are using NEB buffer, use buffer2. digest
the insert and vector for enough time, and make sure your enzymes
are good (i think so from your description). unless the cloned
gene is harmful to the bacteria host, the cloning problem often
come from incomplete digest for such cloning strategy. |
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s***e
发帖数: 911 | 11
什么是hybridization? 是不是ligation(我是生物盲, 别笑, hehe)?
这个可以作些理论估计的:
hybridization时间可以分成几部分: T_d+T_s+T_l
其中T_d是diffusion time, 也就是两条链相遇的平均时间;
这个时间和分子浓度有关系, 大致是这个分子diffuse出分子平均占有体积的时间.
平均占有的体积是浓度的倒数. 随即扩散出一个一个特征距离x, 扩散理论的结果是:
x^2=D*t, D是扩散系数, t是扩散时间. 扩散系数和温度, 溶液粘滞系数, 扩散体的
尺寸有关系. 固定温度和固定溶液下, D和扩散分子的尺寸R成反比: D正比于R^(-1).
于是T_d正比于C^(-2/3)*R, R是链分子的coil size, 它和链长L的关系是:R=Sqrt[L*b],
b代表链的弯曲弹性---b越小, 链越软. 这里的结果对柔软的长链尤其正确.
于是对特定的分子和特定的浓度来说: T_d正比于L^(1/2).
T_s是self diffusion time, 也就是相遇后调整构型使得端对端相吻合的时间;
这个时间大致是分子 |
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r****o
发帖数: 105 | 12 几个小建议：
(1) 去磷酸化不是那么重要。
(2)换新的连接酶尤其是新的BUFFER。连接反应的BUFFER
非常容易坏，因为里面有ATP，冻融几次就不行了。
(3)载体和插入片段的量要足够。建议插入片段的量越多
越好。我一般让片段的莫尔量是载体的几十倍。
(4)既然你用琼脂糖凝胶纯化你的片段。我估计你用UV照射过
胶来确定位置，然后切下有DNA的部分，是吧？UV照射下，
很容易让DNA形成嘧啶二聚体，急剧降低连接效率。所以你要么
不用gel purification，如果必须分离两个片段，你可以
同时在两条LANE上跑少量的DNA和大量的DNA，切下少量DNA的
胶用UV照射确定所要片段的位置。 |
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c*******o
发帖数: 8869 | 13 digestion of PCR product can be very tricky, what i do is following:
1 purify your PCR product with qiagen kit
2 use T4 PNK phosphorylate your PCR product's blunt end
3 ligate your blunt PCR fragment into any vector cutted with blunt end enzyme
(like EcoRV)
4 transformation and mini prep plasmid
5 then use hindIII and EcoRI cut the fragment out of vector
6 purify your hindIII-EcoRI fragment with kit and insert it into the vector
you want to use
this will add one more subcloning step but i am sur |
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p*****n
发帖数: 981 | 14 ☆─────────────────────────────────────☆
jacinta (我不做妖很久了) 于 (Wed Jan 31 11:25:54 2007) 提到:
agar plate + kana 长出了克隆后（但是这些克隆长得很奇怪，偏小，每一个周围都有
很多更小的卫星一样的点点）。挑了一些miniprep，3ml + kana，第二天没有任何细菌
长出来！LB清澈透明！
怎么回事？原来板上的那些是假阳性？我是：Insert DNA + pGL3 (already made
before)---> StuI (blunt end) + NocI (to cut some fragment of insert DNA)-->
Klenow make blunt end--> ligation (recirculation) --> transform
如果没有recirulation的话，是不会有克隆的啊。怎么会是假阳性呢？不是假阳性怎么
解释现在不长呢？
☆─────────────────────────────────────☆
mickoal |
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r******m
发帖数: 173 | 15 I am trying to clone a 300 bp gene into pTYB2 vector (7.4kb)for later
protein purification method by IMPACT-CN. NdeI (producing sticky end) and
SmaI (blunt end) restriction sites were constructed into primers
corresponding to the N- and C- termini of the gene and incorporated by PCR
amplification. The PCR product and the vector were both double digested with
SmaI and NdeI respectively. After gel purification, the digested insert (
gene) and the vector were used to perform ligation with T4 DNA li |
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e****s
发帖数: 1125 | 16 Ligation的时候有没有做Vector的对照？对照盘里面有克隆吗？
PCR
pGEM |
|
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a****d
发帖数: 1919 | 18 Roche dephos and ligation kit works well, try it. |
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x******8
发帖数: 350 | 19 it is sticky end ligation, not blunt end. |
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s*******y
发帖数: 2366 | 20 不了解这个载体，有没有查文献看别人插入最大多大？
可以试着用promega的TSAP (Thermosensitive Alkaline Phosphatase)处理一下单酶切
纯化载体，使其无法self ligate |
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a***a
发帖数: 40617 | 21 你问问题没问对啊
现在你是连接有问题
你是双酶切？酶切是否彻底？是否自连？这些都是问题
你要保证双酶切彻底的情况下用CIP treat以后再用合适的比例进行足够时间的
ligation
克隆问题是板上最常见的问题。我也觉得是最没有必要问的问题
与其这里问，不如问自己lab的师兄、师姐或者postdoc，效果会更好
因为要让别人给你troubleshoot，你至少得写几页的details |
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a***a
发帖数: 40617 | 22 跟你insert多长P关系也没有。。。
唯一的关系就是以后算ligation 摩尔比的时候可以考虑一下 |
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d****h
发帖数: 46 | 23 How do you double digest vector and PCR product with SacI(buffer1 100%,
Buffer3 10%) and SalI(Buffer3 100%, buffer1 0%), sequentially or
simultannously? If you do it simultaneously, both theoretically and
practically(maybe in your case), it won't work.
I made 50 constructs in the past 2 month. In my experience the propotion (of
insert and vector)and ligation condition are not that important, you just
need fragments in good quality.
Never use CIAP unless you have to. |
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J******6
发帖数: 18 | 24 After PCR, do TA cloning first and then cut the inserts out for ligation
with your vector.
It shoud be helpful to treat the vector fragment with CIP beforhand. |
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h*********9
发帖数: 361 | 25 版上时不时就有人上来问克隆问题，我就简单说一下个人的一点点经验：
克隆的第一步一般都是从PCR开始的。PCR关键是引物设计。推荐用免费的primer3。手
动设计注意引物一般GC含量45%-55%。长度在20-24之间。3'端最好不要有很多GC在一起
（40%即可）,以减少非特异产物。内切酶位点一般加在引物的5'端，最好有4-6个以上
保护碱基以利酶切（很多酶对此有硬性要求，详情参见NEB目录）。设计好的引物可以
到www.idtdna.com察看二级结构等引物信息。
第二步酶切（当然也可用TA克隆法）很关键，一定要酶切彻底才会有较高的连接效率，
我的经验是对大多数质粒，一个微克用NEB的酶1微升（10-20 U）/3-4小时，50微升体
系。PCR产物酶量要适度增加因为长度短末端多。难切的酶可酌量延长时间或加大酶量(
难不难切可参见NEB每个酶后面的说明：After a XXX-fold overdigestion with XhoI,
> 95% of the DNA fragments can be ligated with T4 DNA Ligase (at a 5'
term |
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R******E
发帖数: 159 | 26 这个要顶
曾经用过一次磷酸化
结果比不用的self-ligation更高
haohaohao99
方便的话
可不可以帖下
你用的protocol?
量(
XhoI, |
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n********k
发帖数: 2818 | 27 I recall is to both promote the completion of ligation and meanwhile to kill
the ligase... |
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s********s
发帖数: 84 | 28 测基本上是，是把得到的virus稀释后 infect细胞 然后用抗生素blasticidin 选出来
有抗性的以后 染色 数有多少colony。
载体重组了。。如果是这个原因 有啥办法呢。。只能换载体么。。
to neverthink 更多的细节啊
是用PENTR3C （里面有表达目标蛋白的gene）
然后和 plenti6/V5-DEST 做LR recombination
得到colony做酶切 看看位点之类的对不对
最后得到plasmid （用来做transfection之类的能得到蛋白表达）
用这个plasmind然后和pLP1, pLP2, pLP/VSVG之类的一起transfection 293T 细胞
得到virus
然后infection 然后检测蛋白没有。。
曾经用同样的办法做了几批virus，曾经有一批是能检测出来的。
但是后来我再做就不行了= =
反反复复几次以后 老板就让我重做一遍vector 就是从PENTR3C和自己目标蛋白gene 酶
切，ligation那步开始重做。也不说为啥要这样。一摸一样的方法重做一遍会有啥改变
么。。
PENTR3C这个原始的载 |
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O******e
发帖数: 14 | 29 PCR出一段5k genomic DNA，试图克隆进载体。阳性克隆数很低（or 大量假阳性），而
仅有的几个通过clony pcr筛选的克隆，经过酶切或两端测序，发现或者中间缺失，或
者一端缺失，长度在2－3k之间，克隆进去的片段序列正确。Ligation前跑胶确认PCR
insert完整。已困扰3月，请高手解释原因和解决办法。
这段序列含一个基因的3' UTR及之后的大量non-coding genomic DNA，也许克隆后不稳
定？是重组吗？试过几种载体（TA或Blunt）和E.coli，包括Top10，clontech的stellar，还有promega的。
谢谢。 |
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N*o
发帖数: 97 | 30 try stb12 bacteria (grown at 30C); try promoter-less plasmid (pUC18, pUC19).
Or try keeping the insert in two plasmids (e.g. 2kb in one, 3kb in the
other) while cloning and finally ligate the two. good luck! |
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a********k
发帖数: 2273 | 31 最简单的办法，跑完胶把band切下来包在parafilm里面，直接用手把水挤出来。那些
TAE/TBE就可以直接用来做ligation了。效率不高，但是doable |
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X***n
发帖数: 366 | 32 It's more complicate than lentivirus.
Is there any other method than ligated mediate PCR public available yet? |
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n****e
发帖数: 1677 | 33 no kidding, ppl here still enjoy talking about overnihg western, old
subcloning with restriction digestion and ligation... |
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a***e
发帖数: 1010 | 34 I am doing digestion right now, will ligate soon. I am really old. |
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m**i
发帖数: 47 | 35 多谢指教!!因为PCR product之后要做digestion和ligation, 那PCR clean up如果用
kit的话也要好几百个了吧.还是用phenol/chloroform去protein,或者还有什么更方便
快捷的办法去掉taq和dNTP呢? |
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a***e
发帖数: 1010 | 36 (1)try p32 probe, that's better than dig-probe. try use StarFire kit to
increase the sensitivity of your probe (~10x).
(2)40-50 nt probe is more than enough. People can usually blot small RNAs
with 20-30 nt probes (after StarFire labeling)
(3) you can try Tagman-qPCR or splinted-ligation assay, or LNA probe to do
northern blot
(4) deep sequencing w/ or w/o cloning |
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z*********o
发帖数: 1091 | 37 Yes, actually I redid it from ligation step. I really could not understand
why all six clones did not work, but in the meanwhile, the other extracts
from different clones (different construct) worked well. I think the stuff
that I used should be clean. |
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a***e
发帖数: 1010 | 38 you need a bridge DNA.
check NEB
Taq DNA Ligase catalyzes the formation of a phosphodiester bond between
juxtaposed 5′ phosphate and 3′ hydroxyl termini of two adjacent
oligonucleotides which are hybridized to a complementary target DNA. The
ligation will occur only if the oligonucleotides are perfectly paired to the
complementary target DNA and have no gaps between them; therefore, a single
-base substitution can be detected. Taq DNA Ligase is active at elevated
temperatures (45°C-65°C) (1,2). |
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x**2
发帖数: 255 | 39 最近一个克隆做的很郁闷，还望各位高手相助~
谢谢！
由于实验需要，要把两个DNA片断插入到昆虫细胞表达载体(pFastbac)的同一个多克隆
位点中，该表达载体内的多克隆位点有BssHII，EcoR1, Xba1和HindIII。
我要插入的片段是：
EcoR1-片段1-Nhe1（Xba1的同尾酶），和Xba1-片段2-HindIII，
我尝试过三个片段一起连接(three way ligation)，板子上长的两个点都不对（平行做
的BssHII-片段4-Nhe1，Xba1-片段2-HindIII成功得到正确质粒）。
我后来先把片段2插入到载体中，得到质粒：载体-EcoR1-Xba1-片段2-HindIII-载体。
然后再尝试把EcoR1-片段1-Nhe1插入，板子上长的两个点还是都不对，得到的质粒用双
酶切鉴定好像是自连质粒（虽然我双酶切的载体-EcoR1-Xba1-片段2-HindIII-载体）。
个人认为不是双酶切载体的问题，因为：1，EcoR1, Xba1两个酶切位点相隔50bp；如果只
有一个酶切动了质粒那么应该会有很多自连载体，而我的板子上只得到两个点。
我测序鉴定过载体 |
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a***e
发帖数: 1010 | 40 when u order oligo, the end is -OH, you have to kinase first by T4 PNK.
you can kinase (+) & (-) strands together in 50 ul volumn. then boil it
in water, let the water cool down on bench gradulately.
then it is enought for ligation. |
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c*********r
发帖数: 1046 | 41 Thanks a lot!
Do I need to purificate the product of anneal before ligation? |
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X***n
发帖数: 366 | 42 我的做法是：
1. Linearize plasmid with restriction enzymes.
2. 1 uM sense and antisense strand in Qiagen EB buffer, 95oC 5min, turn off PCR machine directly.After half an hour chill it on ice.
3. 10-100 ng linearized plasmid plus 1 uL 1 uM annealed insert, 5 uL fast ligation system, RT. 10 min.
4. Transform DH5alpha competent cell.
I don't think you need to phosphorylation of the insert.It works for me. |
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W*H
发帖数: 3 | 43 I am doing a cloning that involves inserting three large pieces into a
vector. I remembered that in biology Board someone recommended a kind of
ligase that works well for large pieces ligation. However I could not find
it by searching the biology board. Please help me. Your help is greatly
appreciated. |
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S*****s
发帖数: 287 | 44 我正在做一个 5 kb 和 8.2 kb 片段的 ligation，效率蛮高，就是用 NEB T4 ligase
16 度反应过夜，然后用电转化。你做的片段多大？ |
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b******k
发帖数: 1874 | 45 for this topic, many ppl replied. I have compiled some of them here. (btw, s
ome of them are better than the one marked). while majority of ppl suggest t
o add Pi tto oligo 3' end by TNK, I don't know why. Anyone could help give r
elated mechanisms? Thanks a lot.
???: smartkevin (Biofuels), ??: Biology
? ?: Re: ????:?????30bp?DNA fragment ???vector?
???: BBS ????? (Tue May 18 04:01:41 2010, ??)
??oligo?????????????,???????????
??????protocol,??????:
1. oligos?TE or H2O??,??100 uM;
2. ???????,37 |
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b******k
发帖数: 1874 | 46 i will post it again when back home. sorry, sounds my pc in schoool doesnt'
support chinese font.
s
t
r |
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m****r
发帖数: 383 | 47 I got some self-ligation product. Not sure if this can serve as a control. |
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n******d
发帖数: 680 | 48 ligation to T vector first.
反向设计引物扩增，cut掉一部分后直接加入梅切位点，然后连入你的目的片段。
over |
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s******y
发帖数: 28562 | 49 DNA are double helix
On the vector part there will be one 5'-end and one 3'-end on each side.
If only one end has the phosphate group, it will be enough for the ligation
to work. The nicked end can be patched up later inside the bacteria.
In some case, in order to increase the efficiency of transformation, people
use T4 ligase to put the phosphate group on the two 5'-ends of the oligo. I
think this is why your senior in the lab ask you to do that. In this case,
you should follow his/her directio |
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s******y
发帖数: 28562 | 50 4kb is not too long ah.
I used to Pfu Turbo (Strategen) for a gene at that length with GC% =65% and
it works fine. I added 0.1% DMSO in the buffer.
Another suggestion is, you can try to amplify two fragments in the same gene
seperately and then ligate them together later. I found it easier for
tricky sequences. |
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