how many colonies can you get in a electro-tranformation after gibson assembly

[qiansha...@gmail.com]

Hi,

recently, i am making a custom sgRNA library. I ordered Oligos as Nat protocol paper described, and PCR it with Array-F/R primers.

I gel purified 140bp band and assemble it to BsmBI-cutted Lenti-crispr v2 vector for 60min 50 degree incubation. i dilute it for 4 times, and direct electro-transform them into Lucigen competent cells. but i did not get any colonies in

AMP plate.

Can anyone helps me in this experiment and share some tips.

For lucigen competent cells, how much colonies can you get in one electro-transformation?

Thanks!

PS. Gibson assembly reaction is 330ng Vector and 50ng 140bp band.

[Julia Joung]

Hi,

For lucigen competent cells, I typically get at least a few million colonies per electroporation with 100ng of plasmid input. From your description of what you did, it seems like you diluted the Gibson reaction instead of doing an isopropanol purification as the protocol suggested. The salts in the Gibson reaction may reduce your electroporation efficiency, and that may be why you did not get any colonies.

Hope this helps!

Best,

Julia

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[qiansha...@gmail.com]

Thanks Julia for offering help!

I have one question about PE wash in gel extraction. i notice guanidinium is still in DNA after PE once wash and i do not know whether it will cause assmbly fauilure.

在 2017年5月15日星期一 UTC+8上午6:36:16，Julia Joung写道：

Hi,

For lucigen competent cells, I typically get at least a few million colonies per electroporation with 100ng of plasmid input. From your description of what you did, it seems like you diluted the Gibson reaction instead of doing an isopropanol purification as the protocol suggested. The salts in the Gibson reaction may reduce your electroporation efficiency, and that may be why you did not get any colonies.

Hope this helps!

Best,

Julia

On Sat, May 13, 2017 at 7:53 AM, <qiansha...@gmail.com> wrote:

Hi,

recently, i am making a custom sgRNA library. I ordered Oligos as Nat protocol paper described, and PCR it with Array-F/R primers.

I gel purified 140bp band and assemble it to BsmBI-cutted Lenti-crispr v2 vector for 60min 50 degree incubation. i dilute it for 4 times, and direct electro-transform them into Lucigen competent cells. but i did not get any colonies in

AMP plate.

Can anyone helps me in this experiment and share some tips.

For lucigen competent cells, how much colonies can you get in one electro-transformation?

Thanks!

PS. Gibson assembly reaction is 330ng Vector and 50ng 140bp band.

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[Julia Joung]

I use the Zymo gel extraction kits, which require 2 washes, but I'm not sure that guanidinium in the Gibson reaction is really what caused you to have no colonies. Have you tried isopropanol purifying your Gibson to see if you get more colonies?

Best,

Julia

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[qiansha...@gmail.com]

Thanks!

在 2017年5月16日星期二 UTC+8上午9:49:21，Julia Joung写道：

[sunmen...@gmail.com]

Hi, Julia. There was no clones on my plate too. I want try your suggestion-  isopropanol purification . But 100ng DNA was invisible even after 12000 centrifuge, can you show the detail of your protocol of isopropanol purification?

在 2017年5月15日星期一 UTC+8上午6:36:16，Julia Joung写道：

Hi,

For lucigen competent cells, I typically get at least a few million colonies per electroporation with 100ng of plasmid input. From your description of what you did, it seems like you diluted the Gibson reaction instead of doing an isopropanol purification as the protocol suggested. The salts in the Gibson reaction may reduce your electroporation efficiency, and that may be why you did not get any colonies.

Hope this helps!

Best,

Julia

On Sat, May 13, 2017 at 7:53 AM, <qiansha...@gmail.com> wrote:

Hi,

recently, i am making a custom sgRNA library. I ordered Oligos as Nat protocol paper described, and PCR it with Array-F/R primers.

I gel purified 140bp band and assemble it to BsmBI-cutted Lenti-crispr v2 vector for 60min 50 degree incubation. i dilute it for 4 times, and direct electro-transform them into Lucigen competent cells. but i did not get any colonies in

AMP plate.

Can anyone helps me in this experiment and share some tips.

For lucigen competent cells, how much colonies can you get in one electro-transformation?

Thanks!

PS. Gibson assembly reaction is 330ng Vector and 50ng 140bp band.

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[sunmen...@gmail.com]

Thanks!

Mengru

在 2017年7月15日星期六 UTC+8上午10:50:13，sunmen...@gmail.com写道：

[Julia Joung]

Hi Mengru,

The isopropanol purification details can be found in our screening protocol here. Please let me know if you have any questions.

Best,

Julia

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[sunmen...@gmail.com]

Hi Julia

 I tried the isopropanol purification protocol and electroporated 100ng but i only get 12 clonies on plate (not diluted), and I wondering there are only 360 clonies with 10ng positive control(NEB Assembly Kit) after electroporate. How many clonies do you get with positive control and why I only got10 clonies on my plates.

PS: I use LentiCRISPR V2 (15K) as my backbone vector and have sequenced the overlap sequence is right and 140 bp insert also have been checked after ligation with T vector and sequenced. 

when I electroporate plasmid I always electroporate 100 ng library plasmid as control and the efficiency is >109.

My ligation system was strict followed your suggestion (330 ng backbone and 50 ng insert)

The backbone and insert fragment are runed gel and size selected , extraction with Qiagen kit and solve in EB buffer.

I think my key problem is ligation efficiency (Am I right?)  Look forward you reply.

sincerely Mengru

 

在 2017年7月16日星期日 UTC+8上午4:47:02，Julia Joung写道：

[Julia Joung]

Hi Mengru,

I have not tried to electroporate the positive control, but 12 colonies on an undiluted plate is definitely much lower than expected for an 100ng electroporation. Did you follow the library cloning described in our screening protocol exactly?

If you did, then the problem might be the electroporation efficiency. In my experience, the lucigen electrocompetent cells should give >1 million colonies when electroporating 100ng of plasmid library.

Best,

Julia

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[qiansha...@gmail.com]

Hi sunmen,

i meet the same puzzlement as you.

i have use the 10^9 effiency mach1-T1(invitrogen) for transformation instead of lucigen endura competent cells, but i get no colony.

so i tried circular polymerase extension cloning method,maybe this will help you!

best wishes

在 2017年7月23日星期日 UTC+8下午9:39:13，sunmen...@gmail.com写道：

[sunmen...@gmail.com]

Thank you for your reply! 

在 2017年7月31日星期一 UTC+8下午2:57:40，qiansha...@gmail.com写道：

[sunmen...@gmail.com]

Hi Julia 

     

     Thank you for your reply.

    

     Finally I made it after namy failures. It benefits a lot from your advice. 

    

     Best.

     Mengru

在 2017年7月26日星期三 UTC+8下午12:41:01，Julia Joung写道：

[Minoo karimi]

Hi Mengru,

I am strugeling with Gibson assembelly effeicny as well. I would highly apprecite if you could share how you solved the issue and what was the problem in your case?

Thanks,

Minoo

[Ece Çakıroğlu]

Hi, 

How did you solve your problem? I'm trying to clone a custom library (~40000 gRNAs) into lentiguide-puro. I followed the instructions of Nat protocols article by scaling down according to my library size. I used 165 ng plasmid and 25 ng insert for 10 ul Gibson reaction and incubated in 50C for 60 min. I electroporated 2 ul of reaction into Endura electrocompetent cells and got 48 colonies (no dilution). Then I tried isopropanol precipitation and electroporated 60 ng sample and got 30 colonies. What can I do to improve the efficiency? 

Sincerely,

Ece

11 Kasım 2020 Çarşamba tarihinde saat 19:46:15 UTC+3 itibarıyla mino.k...@gmail.com şunları yazdı:

[Julia Joung]

Hi Ece,

I would suggest first to electroporate the control plasmid provided with the Endura electrocompetent cells (or any plasmid that you have around), to see if the low efficiency you are seeing is due to low electroporation or Gibson efficiency. The electroporation can sometimes be a bit tricky to master.

Best,

Julia

To view this discussion on the web visit https://groups.google.com/d/msgid/crispr/03459a6b-d37f-440d-a000-9055b9e668c5n%40googlegroups.com.

[Ece Çakıroğlu]

Hi Julia, 

I electroporated pUC19 plasmid provided with the cells and the efficiency was 10^9. After isopropanol precipitation A260/280 was 1.85 and A260/230 was 1.68. Is 1.68 low to continue with electroporation? Is purity of digested plasmid and insert significant for the Gibson reaction to work efficient? (A260/280 of digested plasmid is 1.83 and A260/230 is 1.97. A260/280 of the insert is 1.69 and A260/230 is 1.55)

Julia Joung <juliaj...@gmail.com>, 22 Kas 2020 Paz, 03:32 tarihinde şunu yazdı:

--

Ece Çakıroğlu, MSc.

Senturk Lab

Izmir International Biomedicine and Genome Institute

Dokuz Eylul University, Health Campus

Inciralti / Izmir

[Bengisu Uluata]

Hello all,

We are struggling with a similar problem in Gibson assembly. We are following the protocol by Joung et al (2017), however using the primer sets proposed in Cold Spring Harbour paper ( doi:10.1101/pdb.prot090803 ), which is shorther in comparison to primer pairs in Joung et al. We have a few questions:

i. Do the primer size and oligo synthesis purification method (MOPC versus HPLC) affect Gibson assembly efficacy?

ii. Do the  purity of backbone as well as insert interfere with the assembly?

Thanks in advance

22 Kasım 2020 Pazar tarihinde saat 12:16:01 UTC+3 itibarıyla ececk...@gmail.com şunları yazdı:

[Julia Joung]

Hi,

To answer your questions:

1. If the overlap region between the insert and vector is shorter, the Gibson assembly may not be as efficient. I haven't tested shorter overlap lengths, so you will have to test to see. We typically use primers without additional purification.

2. Yes the purity of the insert and backbone can affect assembly. Add extra wash steps in the purification if necessary.

Best,

Julia

To view this discussion on the web visit https://groups.google.com/d/msgid/crispr/58f71240-3db7-4aea-945c-5c821d5e991en%40googlegroups.com.