gDNA PCR Optimisation

[Mehmet Hazirci]

Hi everyone, 

Would like to firstly express how useful this forum is in troubleshooting and also understanding more about CRISPR screening - so thank you all for sharing your knowledge and experience! 

I am using the GeCKO v2 library and following the protocol from Joung et al. Nature 2017 with reasonable success!

After extracting gDNA, I will perform the necessary PCR reactions to amplify the library ready for NGS sequencing and analysis of screening results. I want to optimise both gDNA input per reaction, as well as cycle number. 

In this optimisation process, am I just looking for a solitary 260-270bp band in my 2% agarose gel? ie. the most successful condition is that with the brightest band and no other bands apart from the 260-270bp band (I am using the KO library). If not, what am I looking for in order to determine the optimal DNA quantity per reaction and cycle number? 

Thanks in advance for your help. 

Best wishes,

Mehmet

[Julia Joung]

Hi Mehmet,

Thank you for your interest in our work.

The best condition for NGS PCR is the highest ratio of the target PCR product at 260-270bp compared to primer dimer. You will want to aim for the lowest PCR cycle number at which you can see a visible target band on the agarose gel. Usually this should be 22-25 cycles.

Best,

Julia

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[Mehmet Hazirci]

Thanks for your advice Julia!

[Mehmet Hazirci]

Hi Julia,

I've attached some pictures of the results of the optimisation experiments I performed. ug numbers refers to the amount of gDNA used for the PCR reaction, which was then PCR purified with spin column kits and 300ng of purified PCR product was used as input into this gel. I am not getting any primer dimers in any of the samples, however band intensity does appear to decrease after 3.5 ug gDNA. Am I still looking for the brightest band, given no primer dimers, or am I okay to increase gDNA input further? Ideally, I would want to use the highest amount of gDNA possible per PCR reaction as I have lots of gDNA to get through! This is using 21 cycles - I do not think I should increase the cycle number further as there is still a visible band at all amount of gDNA. 

Grateful for your and anyone else's thoughts on this!

Thanks again for your help.

Best,

Mehmet

On Tuesday, May 6, 2025 at 6:13:38 PM UTC+1 juliaj...@gmail.com wrote:

[Mehmet Hazirci]

On Tuesday, May 6, 2025 at 6:13:38 PM UTC+1 juliaj...@gmail.com wrote: