CRISPR Library Amplification

[MikeT]

Hello, 

I am having trouble amplifying the Brunello library in lentiCRISPRv2 from Addgene. We have a major contamination band between 1-1.5kb on DNA gel. Has anyone experienced this?

Thanks!

Mike

[Julia Joung]

Hi Mike,

I have not heard about the potential contamination with the Brunello library. Maybe talk to Addgene about it?

Best,

Julia

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[Gunjan Kumar]

Hi Mike,

Yes, I am aware of this problem! We had the same issues when we amplified the Brunello library (see attached image - I assume yours looks similar). We contacted addgene and they said that expanding pooled libraries sometimes results in amplification of a smaller band as a result of a recombination event. They isolated the DNA from that smaller band, fully sequenced it, and found it to be a recombination product that only contains an ori and the AmpR gene.

They said that this shouldn't be a problem because the DNA from the smaller band doesn’t contain lentiviral packaging sequences or the PuroR gene (so will not be selected for during screening).  I also contacted Dr. Doench for feedback on this 1kb band and he indicated that the presence of that smaller recombination product should not adversely affect library function; however, he did say that this band would get stronger with each subsequent amplification so he recommended using the original pooled library from addgene if you did plan on amplifying the library again in the future. I had asked addgene to updated this information on Brunello library page but they have clearly neglected to do so. 

Hope this helped!

Best,

Gunjan

[shyguy...@gmail.com]

Hi,

We’ve been testing out amplification of Brunello library and have some queries as well...

Here’s a gel photo of GeCKO A vs Brunello library pDNAs after amplification. We’ve also obtained a ~1.5 kb band (seems like a recombination too) and we’re not that sure if we should proceed with this or gel extract it before making the viruses. 

- What conditions do we have to vary to minimize such recombination?

- Has anyone ordered Addgene’s Brunello viruses too? Would like to know how they’re working and type of media used, estimated viability of these viruses and perhaps any precautions to be taken.

- Any comments on their empirical sgRNA distribution would be helpful too

Thanks!

Chris

[Rehab Heikal]

Hi all,

I'm currently using Brunello library as well and I observed a very intense band of the recombinant product (attached image). I see this thread was 2018 , so I hope that your experiment went well.
 I just wanted to check if anyone of you had proceeded with the lentiviral packaging without getting rid of this recombinant  band ? and also,  Do you know if this band contains  the sgRNA sequence or not? I'm asking because if I want to proceed and do NGS on the library to check the sgRNA distribution, will this band affect the NGS reads?

I will really appreciate if someone could answer  my questions and/or give me advises based on your experience with this kind of library. 

Thanks in advance!
Rehab