PCR conditions and gel purification

Jordi Martinez-Quintanilla

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Dec 28, 2020, 9:40:47 AM12/28/20

to Genome Engineering using CRISPR/Cas Systems

Hello,

I have some questions about gDNA PCR after screening.

In Dr. Joung Nature Protocol paper after PCR maxiprep library they performed gel extraction (step 35). However, when they performed the screening and PCR of gDNA they perfomed PCR prufication using the Zymp-Spin V kit (step 59) but not gel extraction. Is it necessary to perform gel extraction of the band?

On the other site, reading some conversations I realized that people mentioned about 2 step PCR but in the paper said perfom only one step PCR, right? I was confused.

Thank you very much for all your help.

Yours,

Jordi

Julia Joung

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Dec 31, 2020, 11:27:31 AM12/31/20

to Jordi Martinez-Quintanilla, Genome Engineering using CRISPR/Cas Systems

Hi Jordi,

Yes we do recommend gel extraction after the PCR of gDNA. The 1-step PCR is for amplifying the sgRNA sequences from the library. The 2-step PCR is for amplifying the target sequences and assessing indel rates during validation of knockout screens.

Best,

Julia

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Jordi Martinez-Quintanilla

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Dec 31, 2020, 11:54:18 AM12/31/20

to Julia Joung, Genome Engineering using CRISPR/Cas Systems

Thank you very much Julia!

Then, shall we perform large-scale PCR purification with Zymo-Spin V columns or we can just run 2ug of the PCR product in a gel and perfom gel extraction? What do you recomend?

Happy New Year.

Best,

Jordi

Julia Joung

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Dec 31, 2020, 12:00:36 PM12/31/20

to Jordi Martinez-Quintanilla, Genome Engineering using CRISPR/Cas Systems

Hi Jordi,

You can pool all of the PCRs together, mix, and PCR purify a smaller fraction of it before gel extraction. It's a bit less work than the large-scale PCR purification, but you want to make sure to purify enough for gel extraction. I prefer doing the PCR purification before gel extraction because the bands run smoother.

Best,

Julia

Jordi Martínez-Quintanilla

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Dec 31, 2020, 1:52:49 PM12/31/20

to Julia Joung, Genome Engineering using CRISPR/Cas Systems

Great information Julia!!

Thank you very much!!!!

Jordi

Sent from my iPhone

On 31 Dec 2020, at 18:00, Julia Joung <juliaj...@gmail.com> wrote:

Varsha Venkatarangan

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Jan 27, 2021, 5:47:34 PM1/27/21

to Jordi Martínez-Quintanilla, Julia Joung, Genome Engineering using CRISPR/Cas Systems

Hi Julia

to follow up on this concern, I did a PCR amplification , PCR clean up, then I loaded 2ug of PCR product on a gel and did a qiaquick gel extraction as per the protocol. I submitted it to NGS and I got a comment saying major portion of the DNA is 440 bp while a small portion is 270 bp. However on the gel, I had a very strong band near 250bp of the marker and almost nothing around 440 bp. What do you think went wrong in this scenario?

Thank you

Varsha

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Julia Joung

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Jan 30, 2021, 12:27:38 PM1/30/21

to Varsha Venkatarangan, Jordi Martínez-Quintanilla, Genome Engineering using CRISPR/Cas Systems

Hi Varsha,

There are a number of posts on this forum related to the same issue. I think your library is most likely fine, with the correct size. If the NGS core used tapestation or bioanalyzer, they will tend to see the larger fragment.