Hello!
I am performing genome-wide CRISPRa. I am following Joung et al. 2017 from Nature protocols. I have performed initial bacterial transformation of whole-genome and maxi prep. Now I need to sequence it. For that I need to prepare library PCR for NSG.
It is not clear the concentration and what primers to order. I think I need to order all 10 forward primers (10 NSG-Lib-Fwd) and 8 reverse primers (NSG-Lib-SAM-Rev). It is my scientific guess that I need to make a mix of all fwd primers (0.5 microL of each in 95 mcroL of water???) and add the mix plus different rev primers per sample. So, it should be 8 samples in the end?
The paper says to add 1.25 microL of rev primer and 1.25 microL forward primer. What primer concentration should I order?
Any information will be helpful!
Thank you,
Tetiana
CRISPR-CAS-9 genes are essential for adaptive immunity in select bacteria and archaea, enabling the organisms to respond to and eliminate invading genetic material. Though, the influential properties of Type II speed the development of eukaryotes. Chiefly gathering of the two type's crRNA and tra-crRNA yields (sgRNA) single guide RNA cemented the pathway of expansion.
Hi! Thank you, this is very helpful. However, I have some questions regarding some technical details about primers. Please see above.
Thank you,
Tetiana
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