GeCKO library amplification, transformation efficiency and maxiprep yield

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.

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Fuchsia Kelpieover 4 years ago

1. Technically speaking for you one electroporation will be enough. I have noticed that for the Endura cells I've been getting a lot more colonies than I used to, so your results are not surprising. Usually for this step, I tend to overshoot the number of electroporations in case you encounter any variability.

2. For 1.1x10^7 colonies, I would go for 3-4 large plates (30-40 standard petri dishes).

3. For the macherey nagel kits, the 0.45g of bacterial pellet per column refers to the dry pellet weight, and when you harvest the bacteria you will measure the wet pellet weight, which may be a lot higher especially if you are not growing a confluent plate. Typically I use 1 maxiprep column for 2 large plates (20 standard plates). I wasn't around during GeCKO, but I definitely use more than 0.45g of bacterial pellet for a macherey nagel maxiprep column. No criteria for a minimum yield, as long as you have sufficient number of colonies and your library QC by NGS looks good.

Seashell Pegasusover 4 years ago

I am really thankful for your kind help. Thanks a lot!

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