P*****s
发帖数: 375 | 1 well, I'm a newer at bichemistry, so just discuss this rather than give
an answer.
I think they just define endonuleases as those -ases that can cleave
DNA at target sites. This is a must for the insertion of intron. So
there seems no way to clone gene without it. (Or you may ask: Can a
gene be cloned without cleaving DNA?) |
p**h
发帖数: 99 | 2 理论上, 有ligase, 就可以克隆 (in vitro). DNA可以用化学法, 超
声等弄断, DNA polymerase 补起, 连接, 筛选. that is just really hard to do.
but not impossible.
【在 P*****s 的大作中提到】
: well, I'm a newer at bichemistry, so just discuss this rather than give
: an answer.
: I think they just define endonuleases as those -ases that can cleave
: DNA at target sites. This is a must for the insertion of intron. So
: there seems no way to clone gene without it. (Or you may ask: Can a
: gene be cloned without cleaving DNA?)
|
p**h
发帖数: 99 | 3 有种鸟枪法(瞧这名字)就是用超声呀, 不是很难的.
另外化学法也很常用.
这些'弄断'方法就是特一性不如没法.
【在 P*****s 的大作中提到】
: well, I'm a newer at bichemistry, so just discuss this rather than give
: an answer.
: I think they just define endonuleases as those -ases that can cleave
: DNA at target sites. This is a must for the insertion of intron. So
: there seems no way to clone gene without it. (Or you may ask: Can a
: gene be cloned without cleaving DNA?)
|
P*****s
发帖数: 375 | 4 化学法本质上和酶法应该是一个原理吧，改变化学性质使某些键松散
但超声不难么？破坏植物等某些细胞时就用了超声法来破碎细胞壁，
那还是蛋白质，DNA应该比蛋白质难弄断的多啊
具体鸟枪法的功率得有多大呢。而且，既然不强调专一性，
那用强激光来弄断应该也可以吧？
我没做过这些实验，全是看书的知识，请指教乐。
【在 p**h 的大作中提到】
: 有种鸟枪法(瞧这名字)就是用超声呀, 不是很难的.
: 另外化学法也很常用.
: 这些'弄断'方法就是特一性不如没法.
|
p**h
发帖数: 99 | 5
本质和原理的说法与你展在哪层次上看问题有关, 它们的一般性作法很不同的.
为什么? 一个脂键, 一个是肽键, 差别应该不是很大吧. 而且是不是肽键更加强以
(因为有点接近双键), 这个是乱猜的.
长的DNA实际上是可以被快速旋转的涡流打断.
没做过, 不知道.
应该可以. 但俺没听说过.
【在 P*****s 的大作中提到】
: 化学法本质上和酶法应该是一个原理吧，改变化学性质使某些键松散
: 但超声不难么？破坏植物等某些细胞时就用了超声法来破碎细胞壁，
: 那还是蛋白质，DNA应该比蛋白质难弄断的多啊
: 具体鸟枪法的功率得有多大呢。而且，既然不强调专一性，
: 那用强激光来弄断应该也可以吧？
: 我没做过这些实验，全是看书的知识，请指教乐。
|
c*****t
发帖数: 1879 | 6 PCR, then use a blunt-end or TA cloning kit (depending on the DNA
polymerase used) to clone it into a plasmid.
To check, pick a colony, stick it to a PCR tube w/ appropriet mix,
use regular PCR cycle w/ slightly longer 95C at beginning to break
up the cells.
Absolutely no endonuclease involved. If using pfu, at most exonuclease
activity is observed.
hoho |
g******k
发帖数: 188 | 7
I forgot one thing, it was suppose to be 1950's, when there is either no PCR
nor ligase, |
p**h
发帖数: 99 | 8 just synthesize one.
【在 g******k 的大作中提到】
:
: I forgot one thing, it was suppose to be 1950's, when there is either no PCR
: nor ligase,
|
p**h
发帖数: 99 | 9
not ligase, syn the DNA. |
c*****t
发帖数: 1879 | 10 Easy, I will just point a region on a bacterial chromosome and
say that's a gene. Unless you could give a precise definition of
gene cloning in 1950s, I wouldn't even bother.
【在 g******k 的大作中提到】
:
: I forgot one thing, it was suppose to be 1950's, when there is either no PCR
: nor ligase,
|
c*****g
发帖数: 1856 | 11 我觉得clone gene don't need to cut the DNA off ba.
i think some ribozyme can work on this.
【在 P*****s 的大作中提到】
: 化学法本质上和酶法应该是一个原理吧，改变化学性质使某些键松散
: 但超声不难么？破坏植物等某些细胞时就用了超声法来破碎细胞壁，
: 那还是蛋白质，DNA应该比蛋白质难弄断的多啊
: 具体鸟枪法的功率得有多大呢。而且，既然不强调专一性，
: 那用强激光来弄断应该也可以吧？
: 我没做过这些实验，全是看书的知识，请指教乐。
|
