Author: Long T. Nguyen; Brianna M Smith; Piyush K. Jain
Title: Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection Document date: 2020_4_14
ID: n5kpvsvg_32
Snippet: In-vitro digestion reactions were carried out with three different types of the Cas12a family (LbCas12a, AsCas12a, and FnCas12a were purchased from New England Biolabs Inc., Integrated DNA Technologies Inc., and abm®, respectively) and a wide array of modified crRNA's (purchased from DNA Technologies Inc.). Cas12a and crRNA were mixed with 1:1 ratio (100 nM:100 nM) in 1X NEBuffer 2.1 and pre-incubated at 25 o C for 15 minutes to promote the ribo.....
Document: In-vitro digestion reactions were carried out with three different types of the Cas12a family (LbCas12a, AsCas12a, and FnCas12a were purchased from New England Biolabs Inc., Integrated DNA Technologies Inc., and abm®, respectively) and a wide array of modified crRNA's (purchased from DNA Technologies Inc.). Cas12a and crRNA were mixed with 1:1 ratio (100 nM:100 nM) in 1X NEBuffer 2.1 and pre-incubated at 25 o C for 15 minutes to promote the ribonucleoprotein complex formation. DNA activator (GFP or PCA3 fragments) (final concentration of 7 nM) was then added to the mixture to produce a total volume of 30 ïL and incubated at 37 o C for 30 minutes. The sample was then analyzed in either 1% agarose gel (for GFP fragments amplified from the pEGFP-C1 plasmid) pre-stained with either SYBER Gold (Invitrogen), GelRed (Biotium Inc.), or premade 15% Novexâ„¢ TBE-Urea Gel (Invitrogen) 31 .
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