u***e
发帖数: 71 | 1 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 |
a****d
发帖数: 1919 | 2 Two step PCR should work for your propose.
synthesize two pair of primers:
F1-xxxx
30bp-xxxx-R1
F2-xxx-30bp
xxxx-R2
PCR separately, then put together both products as template, PCR again using
F1-xxxx and xxx-R2. The final PCR product could be subcloned into the
plasmid. You have to find the suitable digestion sites when designing
primers though. |
H*g
发帖数: 2333 | 3 I will use Quikchange Site-mutagenesis Kit to do this insertion. |
s***m
发帖数: 6197 | 4 22kb会不会太长了？
【在 H*g 的大作中提到】
: I will use Quikchange Site-mutagenesis Kit to do this insertion.
|
I***a
发帖数: 13467 | |
H*g
发帖数: 2333 | 6 The longest I've done is around 15kB and to insert 2 kB, very very tricky
and picked a lot to get the correct one~
Not sure about 22KB, I guess it is worth trying.
【在 s***m 的大作中提到】
: 22kb会不会太长了？
|
s******y
发帖数: 28562 | 7 太长，很难得到中间不出任何错的全长产物。
楼上建议的那个2-step PCR 是正确方案。关键就是要找两个unique restriction
sites用来做Primer F1 and Primer R2, 最后把PCR product 连进去。
【在 s***m 的大作中提到】
: 22kb会不会太长了？
|
z*t
发帖数: 863 | 8 30bp能用的酶切位点不多吧？是不是可以试试平末端连接？
【在 s******y 的大作中提到】
: 太长，很难得到中间不出任何错的全长产物。
: 楼上建议的那个2-step PCR 是正确方案。关键就是要找两个unique restriction
: sites用来做Primer F1 and Primer R2, 最后把PCR product 连进去。
|
s******y
发帖数: 28562 | 9 不是这个意思。
要找的两个酶切位点都是离开要放进去的位置有一点距离的（比方说各离５００bp)
然后把整个长度(1kbp)的终产物给PCR出来，然后再利用那两个位点把全长一千的
产物放回去。
【在 z*t 的大作中提到】
: 30bp能用的酶切位点不多吧？是不是可以试试平末端连接？
|
a****d
发帖数: 1919 | |
|
|
a****k
发帖数: 1130 | 11 这个22kb的特定位置可以切开吗？
【在 u***e 的大作中提到】
: 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
|
X***n
发帖数: 366 | 12 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
【在 u***e 的大作中提到】
: 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
|
f**u
发帖数: 346 | 13 这种情况用ssDNA做recombineering是最好的，然后PCR盲筛。
400到500个colony里面肯定有你要的。
【在 u***e 的大作中提到】
: 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
|
h***e
发帖数: 146 | 14 先用recombineering引入一个counter selection marker, 然后用合成的ssDNA每端大
约50bp的同源臂，再次recombineering消去反向选择的marker，也不用盲晒，酶切鉴定
然后测序。我现在就在做点突变用这个方法，大约20kb，效率在10-20%左右 |
n********k
发帖数: 2818 | 15 Recombination/gateway cloning would be the way to go, very easy if you know
the system, check liu pengtao's work...
For traditional cloning, Sunny's suggestion is the way to go... very easy
too if one is experienced with the MB and the 22kb plasmid itself is not
weird...try to have the insert size about 100-500bp...size in this range are
easier to handle than longer or shorter ones in general...
【在 u***e 的大作中提到】
: 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
|
s******y
发帖数: 28562 | 16 Recombination/gateway cloning 怎么用来做这个么？能不能解释一下？
know
are
【在 n********k 的大作中提到】
: Recombination/gateway cloning would be the way to go, very easy if you know
: the system, check liu pengtao's work...
: For traditional cloning, Sunny's suggestion is the way to go... very easy
: too if one is experienced with the MB and the 22kb plasmid itself is not
: weird...try to have the insert size about 100-500bp...size in this range are
: easier to handle than longer or shorter ones in general...
|
H*g
发帖数: 2333 | 17 Check this out
http://www.genomics.agilent.com/files/LitItems/Absolutely%20RNA
or google
Large Insertions: Two Simple Steps Using QuikChange II Site-Directed
Mutagenesis Kits
【在 u***e 的大作中提到】
: 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
|
z********r
发帖数: 179 | 18 use PCR
recombination would not work because such small fragment will be lost |
n********k
发帖数: 2818 | 19 never did cloning this way myself...but I advised my former roommate to
develop the tech and I remembered he inserted three LoxP sites into a 25kb
or so plasmid within literally 2-3 weeks...I could be wrong since I was not
the one doing it...but I thought the concept was simple: u have the homology
adapters flanking the sequence of interests...someone say 30 bp may be too
small and could get lost...never did myself, don't know(maybe I shouldn't
have commented so surely:)))...I could ask my former roommate...
【在 s******y 的大作中提到】
: Recombination/gateway cloning 怎么用来做这个么？能不能解释一下？
:
: know
: are
|
s******y
发帖数: 28562 | 20 我倒。。。gateway system 不是做这种活的。
用楼上说的，先搞个单链，然后再recombination ，理论上倒是可行，
不过我从来没有那么做过，不知道难度有多大。
你室友具体是怎么做的？去问问他吧，我很好奇呢。
not
homology
too
【在 n********k 的大作中提到】
: never did cloning this way myself...but I advised my former roommate to
: develop the tech and I remembered he inserted three LoxP sites into a 25kb
: or so plasmid within literally 2-3 weeks...I could be wrong since I was not
: the one doing it...but I thought the concept was simple: u have the homology
: adapters flanking the sequence of interests...someone say 30 bp may be too
: small and could get lost...never did myself, don't know(maybe I shouldn't
: have commented so surely:)))...I could ask my former roommate...
|
|
|
a****k
发帖数: 1130 | 21 我是想推荐看看isothermal assembly reaction行不行的。。。
not
homology
too
【在 n********k 的大作中提到】
: never did cloning this way myself...but I advised my former roommate to
: develop the tech and I remembered he inserted three LoxP sites into a 25kb
: or so plasmid within literally 2-3 weeks...I could be wrong since I was not
: the one doing it...but I thought the concept was simple: u have the homology
: adapters flanking the sequence of interests...someone say 30 bp may be too
: small and could get lost...never did myself, don't know(maybe I shouldn't
: have commented so surely:)))...I could ask my former roommate...
|
h******y
发帖数: 55 | 22 在插入位点两侧找合适的酶切位点，然后合成有相同位点的带有30bp插入片段的序列，
直接置换就可以。能找到合适的酶切位点就可以。
大片段backebone连接可以用胶连接。我们做过30多k的。
有没有包子？还没有收过包子呢！ |
a****d
发帖数: 1919 | 23 楼上的，大片段backbone用胶连接，能不能具体解释一下，和ligation有区别吗？ |
d****i
发帖数: 2346 | |
h******y
发帖数: 55 | 25
在低熔点琼脂糖胶里做连接
【在 a****d 的大作中提到】
: 楼上的，大片段backbone用胶连接，能不能具体解释一下，和ligation有区别吗？
|
n********k
发帖数: 2818 | 26 Have u checked Liu pengtao's work? In principle, I don't see why it won.t
work...it is kind of like gateway cloning, but not really, I have to admit
that...
【在 s******y 的大作中提到】
: 我倒。。。gateway system 不是做这种活的。
: 用楼上说的，先搞个单链，然后再recombination ，理论上倒是可行，
: 不过我从来没有那么做过，不知道难度有多大。
: 你室友具体是怎么做的？去问问他吧，我很好奇呢。
:
: not
: homology
: too
|
n********k
发帖数: 2818 | 27 Kind of like add peg, right? Or better?
【在 h******y 的大作中提到】
:
: 在低熔点琼脂糖胶里做连接
|
h******y
发帖数: 55 | 28
没有用过peg这个方法，不知道哪个更好。
【在 n********k 的大作中提到】
: Kind of like add peg, right? Or better?
|
u***e
发帖数: 71 | |
u***e
发帖数: 71 | 30 Thanks. How to link the first two PCR fragments together?
using
【在 a****d 的大作中提到】
: Two step PCR should work for your propose.
: synthesize two pair of primers:
: F1-xxxx
: 30bp-xxxx-R1
: F2-xxx-30bp
: xxxx-R2
: PCR separately, then put together both products as template, PCR again using
: F1-xxxx and xxx-R2. The final PCR product could be subcloned into the
: plasmid. You have to find the suitable digestion sites when designing
: primers though.
|
|
|
n********k
发帖数: 2818 | 31 sure, it is a nice way...nonetheless, sunny's restriction way would be
direct enough...the others could all be nice training/experience but maybe
not neccessary at all...
【在 a****k 的大作中提到】
: 我是想推荐看看isothermal assembly reaction行不行的。。。
:
: not
: homology
: too
|
