b******r
发帖数: 111 | 1 Anyone has experience of inserting 9kb in a 8.4kb vector? 2 months was gone
,but I get nothing. Would
you like to take a look a this protocol and give me some suggestion ?
Thanks a lot
Purpose: Insert 9kb mouse sequence into vector pPNT6.
Vector pPNT6: Its map is at http://www.addgene.org/pgvec1?
f=c&identifier=11072&atqx=pnt6&cmd=findpl . I have inserted 0.9kb DNA in
this vector at XhoI/EcorI
double sites. I should have no problem in cloning short fragment.
Insert DNA is 9kb.I have cloned this 9kb in TOPO vector. I use NotI to cut
this 9kb
from TOPO. The task is to insert this 9kb into pPNT6 at NotI site. My
protocol is:
1. Digest pPNT6 (1 ug),TOPO-9kb(2 ug) with 5 unit of NotI enzyme (NEB) in a
volume
of 50uL,respectively for 3 hours. CIP treats pPNT6 for 1 hour in NotI
digestion mixture.
2. Run the vector(8.4kb) and TOPO-9kb on an 0.7% agarose gel. Make sure
vector and TOPO
-9kb are cut well completely.
3. Use Qiagen gel purification kit to isolate DNA from gel. Run a gel to
estimate the
concentration ratio of vector vs 9kb insert. As compared to DNA ladder, the
purified
vector and 9kb insert should be more than 10ng/uL in 40ul volume. Before
ligation, heat vector and insert
at 65 degree for 10 minutes.
4.Ligation reaction:
9kb insert: 30-150ng;
vector(8.4kb): 10ng;
Ligase:Takara ligase mixture or NEB T4 DNA ligase.
water up to 20 uL
incubate 16 degrees overnight; Didn't do phenol/chloroform extraction,
ethanol
purification for this ligation mixture. DNA were almost lost in previous
phenol/chloroform /ethanol
purification method. PCR using ligation mixture. One primer is T3 from pPNT6
,the other is from 9kb. PCR
result show 9kb is inserted into pPNT6 (false positive?).
6. Transformation protocol:
Heat ligation mixture at 65 degree for 10 minutes. Take 5ul of ligation
mixture to 50 ul DH5 alpha
competent cell placed in ice. Incubate
for 5 minutes,heat shock at 42 degree for 45 seconds,immediately move to ice
and place
for 5 minutes. Add 150 ul of SOC medium,shaking for 1 hour at 250rpm at 37
degree.Take 50ul of
cells on the LB plates contained 75 ug/ml of Ampicilin. Incubate in 37
degree( 30 degree is better for
transformed E.coli to grow?).
7. After 14 hours,some colonies appear. Do colony PCR.One of primers from
vector( T3). The other primer
is from insert 9kb DNA. Picked more than 500 colonies. No positive colony.
The negative control of pPNT6
itself has no colony grown.
8. Isolate plasmid and cut by NotI enzme. Some are empty plasmid,some are
strange plasmid,some has no
plasmid,etc. Anyway,fail to isolate target plasmid.
Any good ligation/transformation kit available for helping troubleshoot for
every step? |
o********r
发帖数: 775 | 2 十年前俺做过这事（17Kb的plasmid），而且过程和你说的很象，最后筛了5个月（每天
就是早上抽质粒，下午PCR/跑胶，然后ligation/transformation，回家前pick colony
/incubation O/N），前后尝试了无数种ligase/competent cell，几乎市面上相关产品
都试了个遍，终于找到一个要的clone。其实17Kb对于某些质粒而言已经差不多是上限
，不稳定，体内很容易发生重组，如果可能尝试不同的vector，或者不同的competent
cell/transformation protocol。另外你或许可以试试Ca++ based
transformation。 |
l****y
发帖数: 398 | 3 buy your officemate a lunch and ask him to try it once for you.
gone
cut
【在 b******r 的大作中提到】
: Anyone has experience of inserting 9kb in a 8.4kb vector? 2 months was gone
: ,but I get nothing. Would
: you like to take a look a this protocol and give me some suggestion ?
: Thanks a lot
: Purpose: Insert 9kb mouse sequence into vector pPNT6.
: Vector pPNT6: Its map is at http://www.addgene.org/pgvec1?
: f=c&identifier=11072&atqx=pnt6&cmd=findpl . I have inserted 0.9kb DNA in
: this vector at XhoI/EcorI
: double sites. I should have no problem in cloning short fragment.
: Insert DNA is 9kb.I have cloned this 9kb in TOPO vector. I use NotI to cut
|
p******i
发帖数: 1092 | 4 you could try XL-10 GOLD competent bugs, for long plasmids.
【在 b******r 的大作中提到】
: Anyone has experience of inserting 9kb in a 8.4kb vector? 2 months was gone
: ,but I get nothing. Would
: you like to take a look a this protocol and give me some suggestion ?
: Thanks a lot
: Purpose: Insert 9kb mouse sequence into vector pPNT6.
: Vector pPNT6: Its map is at http://www.addgene.org/pgvec1?
: f=c&identifier=11072&atqx=pnt6&cmd=findpl . I have inserted 0.9kb DNA in
: this vector at XhoI/EcorI
: double sites. I should have no problem in cloning short fragment.
: Insert DNA is 9kb.I have cloned this 9kb in TOPO vector. I use NotI to cut
|
b******r
发帖数: 111 | 5 Thanks. Frustration around me every day. |
b******r
发帖数: 111 | 6 Why do you think '体内很容易发生重组'?
colony
competent
【在 o********r 的大作中提到】
: 十年前俺做过这事（17Kb的plasmid），而且过程和你说的很象，最后筛了5个月（每天
: 就是早上抽质粒，下午PCR/跑胶，然后ligation/transformation，回家前pick colony
: /incubation O/N），前后尝试了无数种ligase/competent cell，几乎市面上相关产品
: 都试了个遍，终于找到一个要的clone。其实17Kb对于某些质粒而言已经差不多是上限
: ，不稳定，体内很容易发生重组，如果可能尝试不同的vector，或者不同的competent
: cell/transformation protocol。另外你或许可以试试Ca++ based
: transformation。
|
o********r
发帖数: 775 | 7 PCR产物size不对，sequencing证实里面的序列并非是plasmid或者是任何插入plasmid
的序列
【在 b******r 的大作中提到】
: Why do you think '体内很容易发生重组'?
:
: colony
: competent
|
p******i
发帖数: 1092 | 8 加油
【在 b******r 的大作中提到】
: Thanks. Frustration around me every day.
|
r****r
发帖数: 379 | 9 一般大片段克隆，会导致连接效率很低，克隆数大大减少，可以看看你的克隆数和你常做的克隆比是不是还比较正常，如果比较正常，那说明这种假阳性很可能是因为你的插入片段有一段序列比较容易发生重组，试着30度长克隆和摇菌看看。另外，能不用单酶切做克隆就不要用，你大片段克隆还用这个策略，最好换其它双酶切。
另外，单就大片段克隆而言，建议用GeneHogs菌株，我手上的片段普遍10k以上，用这个菌株，从来没出现过问题，感受态效率一般7次方就可以了，15kb可以长百来个克隆。
因此建议先更换单酶切策略和换genehogs，然后不行再换30度生长看。30度生长，已发生重组的质粒，一般10个有2个总是对的。
gone
cut
【在 b******r 的大作中提到】
: Anyone has experience of inserting 9kb in a 8.4kb vector? 2 months was gone
: ,but I get nothing. Would
: you like to take a look a this protocol and give me some suggestion ?
: Thanks a lot
: Purpose: Insert 9kb mouse sequence into vector pPNT6.
: Vector pPNT6: Its map is at http://www.addgene.org/pgvec1?
: f=c&identifier=11072&atqx=pnt6&cmd=findpl . I have inserted 0.9kb DNA in
: this vector at XhoI/EcorI
: double sites. I should have no problem in cloning short fragment.
: Insert DNA is 9kb.I have cloned this 9kb in TOPO vector. I use NotI to cut
|
b******r
发帖数: 111 | 10 Good idea. My 9kb DNA itself contains too many restriction sites. NotI is
the only choice.
'Description:
Important: GeneHogs® competent cells are being discontinued on December
31, 2007.
Use ElectroMAX™ DH10B™ T1R cells for exceptional performance!
'
常做的克隆比是不是还比较正常，如
果比较正常，那说明这种假阳性很可能是因为你的插入片段有一段序列比较容易发生重
组，试着30度长克隆和摇菌看看。
另外，能不用单酶切做克隆就不要用，你大片段克隆还用这个策略，最好换其它双酶切。
这个菌株，从来没出现过问题，感
受态效率一般7次方就可以了，15kb可以长百来个克隆。
发生重组的质粒，一般10个有2个
总是对的。
【在 r****r 的大作中提到】
: 一般大片段克隆，会导致连接效率很低，克隆数大大减少，可以看看你的克隆数和你常做的克隆比是不是还比较正常，如果比较正常，那说明这种假阳性很可能是因为你的插入片段有一段序列比较容易发生重组，试着30度长克隆和摇菌看看。另外，能不用单酶切做克隆就不要用，你大片段克隆还用这个策略，最好换其它双酶切。
: 另外，单就大片段克隆而言，建议用GeneHogs菌株，我手上的片段普遍10k以上，用这个菌株，从来没出现过问题，感受态效率一般7次方就可以了，15kb可以长百来个克隆。
: 因此建议先更换单酶切策略和换genehogs，然后不行再换30度生长看。30度生长，已发生重组的质粒，一般10个有2个总是对的。
:
: gone
: cut
|
r****r
发帖数: 379 | 11 我宁愿改造载体的酶切位点，都不会去做9kb的CIP克隆
December
【在 b******r 的大作中提到】
: Good idea. My 9kb DNA itself contains too many restriction sites. NotI is
: the only choice.
: 'Description:
: Important: GeneHogs® competent cells are being discontinued on December
: 31, 2007.
: Use ElectroMAX™ DH10B™ T1R cells for exceptional performance!
: '
:
: 常做的克隆比是不是还比较正常，如
: 果比较正常，那说明这种假阳性很可能是因为你的插入片段有一段序列比较容易发生重
|
