GeCKO v2 library amplification

[Amoyar Dever]

Hello all,

I will be starting to amplify add gene supplied GeCKO v2 library in next week. I've following couple of questions regarding the library/amplification-

Q1: How many copies of single sgRNA are there in addgene supplied GeCKO v2 human/mouse pooled library? 

Q2: What is the logic behind using 1 electroporation per 10,000 while amplifying the addgene supplied GeCKO v2 library? How the number 10,000 was determined?

[Julia Joung]

Hi Amoyar,

To answer your questions:

1. This depends on which sgRNA, because the relative amount of each sgRNA falls under a normal distribution. You can estimate the average copy of each sgRNA using the size of the plasmid (this depends on whether you use the 1-vector or 2-vector system).

2. From our experience, each electroporation will produce 1-5 million colonies, so the estimated required 1 electroporation per 10,000 sgRNAs in the library should maintain the sgRNA coverage during library amplification at >100.

Best,

Julia

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[Amoyar Dever]

Thank you very much Julia..!

*********************

On Friday, June 8, 2018 at 6:53:33 PM UTC-4, Julia Joung wrote:

Hi Amoyar,

To answer your questions:

1. This depends on which sgRNA, because the relative amount of each sgRNA falls under a normal distribution. You can estimate the average copy of each sgRNA using the size of the plasmid (this depends on whether you use the 1-vector or 2-vector system).

2. From our experience, each electroporation will produce 1-5 million colonies, so the estimated required 1 electroporation per 10,000 sgRNAs in the library should maintain the sgRNA coverage during library amplification at >100.

Best,

Julia

On Thu, Jun 7, 2018 at 10:52 AM, Amoyar Dever <amoyar...@gmail.com> wrote:

Hello all,

I will be starting to amplify add gene supplied GeCKO v2 library in next week. I've following couple of questions regarding the library/amplification-

Q1: How many copies of single sgRNA are there in addgene supplied GeCKO v2 human/mouse pooled library? 

Q2: What is the logic behind using 1 electroporation per 10,000 while amplifying the addgene supplied GeCKO v2 library? How the number 10,000 was determined?

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[Eunkyeong Kim]

Hi Julia,

I posted my own questions about GeCKO library amplification, but I couldn't have answers from anyone. So if you are possible, please help me. Any tips or answers would be really appreciated.

Below is copy of my posting.

I am preparing mouse GeCKO knockout screen. I received two pooled half-libraries from Addgene, and conducted pilot amplification and maxiprep test to ensure the flow of all planned processes.

According to GeCKO paper(2017, nature protocols) 1 electroporation is needed per 10,000 sgRNAs in the library to get more than 100 colonies per sgRNA. Since there are about 65,000 sgRNAs in one half-library, I need to get more than 6.5x10^6 colonies through 6 electroporations. But I obtained 1.1x10^7 colonies through a single electroporation of a half-library in pilot test(I used Endura cells.). I plated electroporated cells on 10 strandard 10 cm petridishes. The efficiency of the transformation was so high that when the cells were plated onto medium, colonies were not formed and looked like carpets. I heard that if cells are plated like a carpet, bias can occur due to inter-colony competition. And lastly, I did maxiprep using the Macherey Nagel kits. I extracted plasmid from ~2.25g of a bacterial pellet according to maxiprep kit manual, and yield was about 290ug (360 ng/ul, 800ul). I'm not sure this yield is enough. And also I found that GeCKO library manual recommend 0.45 g of bacterial pellet per column. So I have three questions now.

1. I obtained 1.1x10^7 colonies, which is more than 6.5x10^6, through a single electroporation. Do I need to electroporate several times(6 times according to paper)? or a single electroporation is enough?

2. I obtained confluent plates. How many plates are needed to avoid carpet-like plate? The GeCKO manual seems to recommend 20 x 10 cm for 6.5x10^6. Then, are 40 plates enough for 1.1x10^7?

3. What is the average yield for 0.45 g of bacteria pellet? Is there a criterion for minimum yield?

Any answers or tips can help me.

Thank you.

Best,

Eunkyeong

2018년 6월 9일 토요일 오전 7시 53분 33초 UTC+9, Julia Joung 님의 말:

Hi Amoyar,

To answer your questions:

1. This depends on which sgRNA, because the relative amount of each sgRNA falls under a normal distribution. You can estimate the average copy of each sgRNA using the size of the plasmid (this depends on whether you use the 1-vector or 2-vector system).

2. From our experience, each electroporation will produce 1-5 million colonies, so the estimated required 1 electroporation per 10,000 sgRNAs in the library should maintain the sgRNA coverage during library amplification at >100.

Best,

Julia

On Thu, Jun 7, 2018 at 10:52 AM, Amoyar Dever <amoyar...@gmail.com> wrote:

Hello all,

I will be starting to amplify add gene supplied GeCKO v2 library in next week. I've following couple of questions regarding the library/amplification-

Q1: How many copies of single sgRNA are there in addgene supplied GeCKO v2 human/mouse pooled library? 

Q2: What is the logic behind using 1 electroporation per 10,000 while amplifying the addgene supplied GeCKO v2 library? How the number 10,000 was determined?

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[Julia Joung]

Hi Eunkyeong,

Please see my response to your independent post - sorry for the delay in response!

Best,

Julia

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