The secondary structure of the target mRNA can be predicted using the RNAfold web server 2 or other online tools. Since the selection parameters of the sgRNA target regions are similar to RNA interference, small interfering RNA (siRNA) designed web tools can be used for sgRNA selection. Three sgRNA/pre-sgRNAs targeting human VEGFA mRNA were designed for the study. The first VEGFA sgRNA/pre-sgRNA was designed based on the targeting sequence of bevasiranib, the first VEGFA-targeted siRNA to reach phase III clinical trial ( Garba and Mousa, 2010). #GRAPHPAD PRISM 8 STEP BY STEP EXAMPLE TRIAL#
The other two sgRNA/pre-sgRNAs were manually designed to target the different regions in VEGFA mRNA, avoiding the stem–loop.
(i) Ten microliters of the ligation reaction or 2 μl of the assembly reaction was added into 50 μl of NEB Stable Competent E.In addition, short fragments of CasRx sgRNA expression cassette (gBlocks TM), CasRx pre-sgRNA expression plasmid, and all-in-one CasRx AAV plasmid were designed and illustrated using Benchling 3. (ii) The mixture was placed on ice for 30 min and heat-shocked at 42☌ for 30 s before being placed on ice for 5 min.Eppendorf tubes were flicked five times to mix the cells with the plasmid DNA. (iv) One hundred microliters of the mixture was spread on carbenicillin (100 μg/ml)-containing agar plates.(iii) Of the NEB Stable outgrowth medium, 450 μl was added into the mixture and the tube was rotated at 37☌ for 1 h.
#GRAPHPAD PRISM 8 STEP BY STEP EXAMPLE MANUAL#.
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