Re: Ng-Ago 重复进展（集合贴）# Biology - 生物学
y*7
1 楼
请问一下有经验的朋友：
网上查了i140 7月13号NSC批了，但是一直没有收到书面的Approval notice。大家一般
几天收到通知？着急！谢谢
网上查了i140 7月13号NSC批了，但是一直没有收到书面的Approval notice。大家一般
几天收到通知？着急！谢谢
H*S
2 楼
I just got some information from the "NgAgo - Google Groups" forum regarding
NgAgo follow-up experiments, Please click the following link:
https://groups.google.com/forum/#!topic/crispr/V1FALNbspCo
NgAgo follow-up experiments, Please click the following link:
https://groups.google.com/forum/#!topic/crispr/V1FALNbspCo
d*3
3 楼
一般一两周最多了吧， 你可以打电话问问，
H*S
4 楼
For those who can not access the link
I copied the content here:
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
NgAgo
15 posts by 9 authors
Davide Seruggia
Jun 23
Hi,
I tried a couple of transfections with the NgAgo plasmid from Addgene and
the 5'P oligo against GFP from the paper, but I did not see a dramatic
reduction in GFP+ as seen using Cas9 and a GFP sgRNA.
Anybody tried as well and came to the same (dead) end?
Davide
k*****[email protected]
Jun 24
Hi, Davide,
I tried the same plasmid that you used to target DYRK1A, HBA2, GATA4, which
are tested in Han's paper, but I didn't see any indel as they did. It seems
there is something wrong with this system.
Kyle
在 2016年6月23日星期四 UTC-7下午6:18:50，Davide Seruggia写道：
- show quoted text -
Jan Winter
Jun 28
Hi guys,
Just had a couple of tests with ngago from addgene and custom Oligos, so I
did not try to reproduce the paper.
And in my hands I see an effect that is slightly less than using the same
target sequence region using the px459, but it works.
I phosphorylated the oligos using T4PNK and transferred 300 ngago per 24
well to hek293t cells.
Cheers
Jan
Haiyang Yu
Jun 28
Hi, Jan,
It is exciting that you got it work! Could you post a detailed protocol?
Please include how you did T4PNK, after that did you purify your oligos? How
did you transfect and how long did you wait?
Thank you!
- show quoted text -
Debojyoti Chakraborty
Jun 28
Hi guys,
We have used this system and it works. We have followed the same protocol as
mentioned in the paper and were able to confirm knockout both on FACS as
well as western. We purified the oligos on gel after T4 PNK and saw the
phenotype after 48 and 72 h.
cheers
debo
- show quoted text -
Debojyoti Chakraborty
Jun 28
just a small correction: we did not purify the oligos. We used 700 ng ngAgo
and 500ng sgDNA for Lipo3000 mediated transfection.
- show quoted text -
z*******[email protected]
Jun 29
Hi debo,
can you give me your protocol purified the oligo on gel, I want to
repeat this experiment, I can't repeat paper of Han, maybe my oligo has some
problems, I order phosphorylation oligo from company。 I can't confirm the
key point whether in here。
在 2016年6月29日星期三 UTC+8下午1:04:05，Debojyoti Chakraborty写道：
- show quoted text -
z*******[email protected]
Jun 29
I want to know，do you have sequencing result?
在 2016年6月29日星期三 UTC+8下午1:19:45，Debojyoti Chakraborty写道：
- show quoted text -
k*****[email protected]
Jun 29
Hi, Jan,
Good to hear that! I ordered the 5' phosphorylated oligo from IDT directly,
and tested DYRK1A, HBA2, GATA4, but it didn't work. I will try your method
to see whether it is the problem of oligos.
Thanks!
Kyle
在 2016年6月28日星期二 UTC-7下午2:34:08，Jan Winter写道：
- show quoted text -
SL
Jun 30
Plasmid GFP expression reduction by NgAgo co transfection is reproducible
but this cannot be confirmed by sequencing. Many factors affect GFP
expression. So far many labs have failed to repeat the genomic DNA targeting
as shown by the paper，which successfully targeted over 50 loci with 100%
success rate and 20% more efficiency. If anyone has obtained NgAho induced
genomic DNA indels confirmed by sequencing please share your data and tricks.
r********[email protected]
Jun 30
I tried in NgAgo in CHO-K1 cells, and after selection with hygromycin for 2
weeks, I got many clonies, and I extracted genomic DNA and did junction PCR,
got a weak band at desired size at both ends. I am trying single clones and
see if I could get better results.
On Thursday, June 30, 2016 at 6:46:27 AM UTC-7, SL wrote:
Plasmid GFP expression reduction by NgAgo co transfection is reproducible
but this cannot be confirmed by sequencing. Many factors affect GFP
expression. So far many labs have failed to repeat the genomic DNA targeting
as shown by the paper，which successfully targeted over 50 loci with 100%
success rate and 20% more efficiency. If anyone has obtained NgAho induced
genomic DNA indels confirmed by sequencing please share your data and tricks.
Jan Winter
Jun 30
Hi guys,
okay some more stuff from my side, a detailed protocol will come once I did
some more tests, so fare I have NO INDEL DATA.
What I did:
- 24 bp oligos
- phosphorylated them using T4 PNK (standard reaction, 300 pM of oligo) for
1 hr at 37 with T4DNA Ligase buffer
- cleaned up oligos using Qiagens Nulceotide removal kit for 4x300 pM of
oligo per column
- measured with Qubit ssDNA kit (but I have no clue about the
phosphorylation)
- transfection of 50000HEK293T cells (24 well plate, 0.5 mL per well, 50000
cells per well, 2 uL TransIT LT1 reagent) with Mirrus TransIT LT1-> 500 ng
of ngAGO from Addgene and 300 ng of oligo
- I do not change any medium, but I add another mL of complete grwoth medium
24 hr after transfection
- FACS assay after 72 to 96 hr
My HEK cells contain a custom reporter as well as Cas9 expression, which is
linked with eGFP by a P2A sequence.
My observations were the following:
- in presence of ngAGO transfection I see a slight reduction of eGFP (even
without any oligo) in the range of 3-5 %
- I see no effect if I transfect 24 bp oligos alone without ngAgo
- I made custom oligos against specific regions of my reporter and target
genes of my reporter systems -> I see an effect in FACS wich is only present
if I transfect ngAgo + Oligos together
- The effect is slightly worse than transfection my sgRNA plasmid (cells are
Cas9 positive)
- If I compare the phenotypic effect of sgRNA vs ngAGO oligos (that target
the same region), ngAgo is less effective
I will do some tests next week and will also have a look at indel formation
in the next month.
Debojyoti Chakraborty
Jun 30
Other recipients: z*******[email protected]
Here's the protocol we followed:
We first phosphorylated the gDNA using the PNK kit from Ambion for 1 hr
followed by heat inactivation at 95 degrees for 5 min. We then went on to
transfect 100,000 cells in 12 well plates as follows:
Mix 1:
Lipo reagent- 3ul
Optimem- 43ul
EGFP N1- 1 ug
NgAgo- 900 ng
gDNA- 500 ng
Mix 2:
Lipo 3000- 2ul
Optimem- 48ul
Mix 3:
Media- 250ul
Optimem- 150ul
Mix1 and Mix2 was incubated for 10min each and then both were mixed together
and incubated at RT for 30min
This was now added to cells. To each well, 50 ul of Mix 3 was added on top.
No further media was added to the cells.
After 12h, the mix was removed and fresh media was added.
- show quoted text -
- show quoted text -
I copied the content here:
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
NgAgo
15 posts by 9 authors
Davide Seruggia
Jun 23
Hi,
I tried a couple of transfections with the NgAgo plasmid from Addgene and
the 5'P oligo against GFP from the paper, but I did not see a dramatic
reduction in GFP+ as seen using Cas9 and a GFP sgRNA.
Anybody tried as well and came to the same (dead) end?
Davide
k*****[email protected]
Jun 24
Hi, Davide,
I tried the same plasmid that you used to target DYRK1A, HBA2, GATA4, which
are tested in Han's paper, but I didn't see any indel as they did. It seems
there is something wrong with this system.
Kyle
在 2016年6月23日星期四 UTC-7下午6:18:50，Davide Seruggia写道：
- show quoted text -
Jan Winter
Jun 28
Hi guys,
Just had a couple of tests with ngago from addgene and custom Oligos, so I
did not try to reproduce the paper.
And in my hands I see an effect that is slightly less than using the same
target sequence region using the px459, but it works.
I phosphorylated the oligos using T4PNK and transferred 300 ngago per 24
well to hek293t cells.
Cheers
Jan
Haiyang Yu
Jun 28
Hi, Jan,
It is exciting that you got it work! Could you post a detailed protocol?
Please include how you did T4PNK, after that did you purify your oligos? How
did you transfect and how long did you wait?
Thank you!
- show quoted text -
Debojyoti Chakraborty
Jun 28
Hi guys,
We have used this system and it works. We have followed the same protocol as
mentioned in the paper and were able to confirm knockout both on FACS as
well as western. We purified the oligos on gel after T4 PNK and saw the
phenotype after 48 and 72 h.
cheers
debo
- show quoted text -
Debojyoti Chakraborty
Jun 28
just a small correction: we did not purify the oligos. We used 700 ng ngAgo
and 500ng sgDNA for Lipo3000 mediated transfection.
- show quoted text -
z*******[email protected]
Jun 29
Hi debo,
can you give me your protocol purified the oligo on gel, I want to
repeat this experiment, I can't repeat paper of Han, maybe my oligo has some
problems, I order phosphorylation oligo from company。 I can't confirm the
key point whether in here。
在 2016年6月29日星期三 UTC+8下午1:04:05，Debojyoti Chakraborty写道：
- show quoted text -
z*******[email protected]
Jun 29
I want to know，do you have sequencing result?
在 2016年6月29日星期三 UTC+8下午1:19:45，Debojyoti Chakraborty写道：
- show quoted text -
k*****[email protected]
Jun 29
Hi, Jan,
Good to hear that! I ordered the 5' phosphorylated oligo from IDT directly,
and tested DYRK1A, HBA2, GATA4, but it didn't work. I will try your method
to see whether it is the problem of oligos.
Thanks!
Kyle
在 2016年6月28日星期二 UTC-7下午2:34:08，Jan Winter写道：
- show quoted text -
SL
Jun 30
Plasmid GFP expression reduction by NgAgo co transfection is reproducible
but this cannot be confirmed by sequencing. Many factors affect GFP
expression. So far many labs have failed to repeat the genomic DNA targeting
as shown by the paper，which successfully targeted over 50 loci with 100%
success rate and 20% more efficiency. If anyone has obtained NgAho induced
genomic DNA indels confirmed by sequencing please share your data and tricks.
r********[email protected]
Jun 30
I tried in NgAgo in CHO-K1 cells, and after selection with hygromycin for 2
weeks, I got many clonies, and I extracted genomic DNA and did junction PCR,
got a weak band at desired size at both ends. I am trying single clones and
see if I could get better results.
On Thursday, June 30, 2016 at 6:46:27 AM UTC-7, SL wrote:
Plasmid GFP expression reduction by NgAgo co transfection is reproducible
but this cannot be confirmed by sequencing. Many factors affect GFP
expression. So far many labs have failed to repeat the genomic DNA targeting
as shown by the paper，which successfully targeted over 50 loci with 100%
success rate and 20% more efficiency. If anyone has obtained NgAho induced
genomic DNA indels confirmed by sequencing please share your data and tricks.
Jan Winter
Jun 30
Hi guys,
okay some more stuff from my side, a detailed protocol will come once I did
some more tests, so fare I have NO INDEL DATA.
What I did:
- 24 bp oligos
- phosphorylated them using T4 PNK (standard reaction, 300 pM of oligo) for
1 hr at 37 with T4DNA Ligase buffer
- cleaned up oligos using Qiagens Nulceotide removal kit for 4x300 pM of
oligo per column
- measured with Qubit ssDNA kit (but I have no clue about the
phosphorylation)
- transfection of 50000HEK293T cells (24 well plate, 0.5 mL per well, 50000
cells per well, 2 uL TransIT LT1 reagent) with Mirrus TransIT LT1-> 500 ng
of ngAGO from Addgene and 300 ng of oligo
- I do not change any medium, but I add another mL of complete grwoth medium
24 hr after transfection
- FACS assay after 72 to 96 hr
My HEK cells contain a custom reporter as well as Cas9 expression, which is
linked with eGFP by a P2A sequence.
My observations were the following:
- in presence of ngAGO transfection I see a slight reduction of eGFP (even
without any oligo) in the range of 3-5 %
- I see no effect if I transfect 24 bp oligos alone without ngAgo
- I made custom oligos against specific regions of my reporter and target
genes of my reporter systems -> I see an effect in FACS wich is only present
if I transfect ngAgo + Oligos together
- The effect is slightly worse than transfection my sgRNA plasmid (cells are
Cas9 positive)
- If I compare the phenotypic effect of sgRNA vs ngAGO oligos (that target
the same region), ngAgo is less effective
I will do some tests next week and will also have a look at indel formation
in the next month.
Debojyoti Chakraborty
Jun 30
Other recipients: z*******[email protected]
Here's the protocol we followed:
We first phosphorylated the gDNA using the PNK kit from Ambion for 1 hr
followed by heat inactivation at 95 degrees for 5 min. We then went on to
transfect 100,000 cells in 12 well plates as follows:
Mix 1:
Lipo reagent- 3ul
Optimem- 43ul
EGFP N1- 1 ug
NgAgo- 900 ng
gDNA- 500 ng
Mix 2:
Lipo 3000- 2ul
Optimem- 48ul
Mix 3:
Media- 250ul
Optimem- 150ul
Mix1 and Mix2 was incubated for 10min each and then both were mixed together
and incubated at RT for 30min
This was now added to cells. To each well, 50 ul of Mix 3 was added on top.
No further media was added to the cells.
After 12h, the mix was removed and fresh media was added.
- show quoted text -
- show quoted text -
y*7
5 楼
谢谢，最多有人等了多久？
因为是公司申请的，自己无法打电话到USICS。还有什么方法？
因为是公司申请的，自己无法打电话到USICS。还有什么方法？
H*S
6 楼
In addition to Addgene, another sources of NgAgo construct from China:
pCDNA3.1-NLS-NgAgo哺乳细胞基因编辑基因敲除质粒合
http://miaolingbio.com/cn/product/plasmid/1826.html
pCDNA3.1-NLS-NgAgo哺乳细胞基因编辑基因敲除质粒合
http://miaolingbio.com/cn/product/plasmid/1826.html
d*3
7 楼
是不是寄给你律师了？
【在 y*******7 的大作中提到】
: 谢谢，最多有人等了多久？
: 因为是公司申请的，自己无法打电话到USICS。还有什么方法？
【在 y*******7 的大作中提到】
: 谢谢，最多有人等了多久？
: 因为是公司申请的，自己无法打电话到USICS。还有什么方法？
y*7
8 楼
我们连Receive Notice（7月6号的）都没有收到，还是通过Check背后的Receipt
number 查到I140 pp 批了。
查过I140材料上的公司联系人地址，绝对没错。
NSC是不是最近不靠谱？
number 查到I140 pp 批了。
查过I140材料上的公司联系人地址，绝对没错。
NSC是不是最近不靠谱？
y*7
9 楼
材料其实是自己准备的，只用了公司的联系人。唉，可恶的USICS！
d*3
10 楼
这个都不清楚了， 打电话问吧， 最坏就是寄丢了吧，
【在 y*******7 的大作中提到】
: 我们连Receive Notice（7月6号的）都没有收到，还是通过Check背后的Receipt
: number 查到I140 pp 批了。
: 查过I140材料上的公司联系人地址，绝对没错。
: NSC是不是最近不靠谱？
【在 y*******7 的大作中提到】
: 我们连Receive Notice（7月6号的）都没有收到，还是通过Check背后的Receipt
: number 查到I140 pp 批了。
: 查过I140材料上的公司联系人地址，绝对没错。
: NSC是不是最近不靠谱？
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