The reaction mix for Cas13 activity was prepared by combining 8.6 μl of nuclease-free water, 2 μl of cleavage buffer (400 mM Tris pH 7.4), 2 μl of LwaCas13a protein diluted in Storage Buffer (SB) to a concentration of 126.6 μg/ml, 1 μl of crRNA (40 ng/μl), 1 μl of the fluorescent reporter (IDT, diluted in water to a final concentration of 4 μM), 1 μl of Murine RNase inhibitor (NEB, #M0314), 0.8 μl of rNTP solution mix (25 mM each, NEB, #N0466), 0.6 μl of NxGen T7 RNA Polymerase (Lucigen, #30223-2) and 1 μl of MgCl2 (120 mM). 2 μl of the RT-RPA-amplified product were then added to the mix. The 20μl-LwaCas13a reactions were transferred in 5μl-replicates (4 wells each sample) to a 384-well, round, black-well, clear-bottom plate (Corning, #3544). The plate was briefly spun down at 500 g for 15 sec to remove potential bubbles and placed into a preheated GloMax® Explorer plate reader (Promega) at 37 °C. Fluorescence was measured every 5 min for 3 h. Data analysis, if not otherwise stated, was performed at the 30-min time-point. The reporter sequence is provided in Supplementary Table 6 .
M0314
Manufactured by New England Biolabs
M0314 is a DNA ligase enzyme that catalyzes the formation of a phosphodiester bond between the 3' hydroxyl and 5' phosphate termini of double-stranded DNA or RNA, thereby joining DNA fragments. This enzyme is commonly used in molecular biology applications such as DNA cloning and ligation.
Automatically generated - may contain errors
5 protocols using m0314
1
Cas13 Activity Assay Optimization
2
Characterization of mRNA Cap Structure
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Commercially-available capped eGFP mRNA (Trilink, CleanCap EGFP mRNA L-7601) at 0.2 uM was exposed to 1 uM Dbr1, 100 U mRNA decapping enzyme (DCE, NEB M0608S), or 200 U yeast scavenger decapping enzyme (yDcpS, NEB M0463S), and incubated at 37 °C for 2 hours. A 10 uL aliquot of the digestion was diluted into 1 ml of TBS supplemented with 1 U/uL murine RNAse inhibitor (NEB M0314), and 150 ug/ml GlycoBlue glycogen (Thermo Fisher Scientific, AM9516), and then 0.5 ml was incubated with 50 uL of anti-2,2,7 trimethylguanosine agarose (Millipore NA02A) for 2 hours at 4 °C with rotation. The immunodepleted flow-through sample was removed, and the beads were washed 3 times by centrifugation. Bound mRNA was eluted by incubation with 1 mM of 7-methylguanosine cap analog (Thermo Fisher Scientific AM8048). Input, flow-through, and elution samples were precipitated with ethanol, and resuspended in 50 uL of 2X RNA loading dye (NEB B0363), separated on a 6% urea-TBE gel (Thermo Fisher Scientific EC6265), and stained with Syber Gold (Thermo Fisher Scientific S11494) and visualized with a UV-transilluminator.
3
Immunoprecipitation of m6A-Containing RNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The remaining fragmented RNA was resuspended in 500 µL Binding/Low Salt Buffer (50 mM Tris-HCl pH 7.4, 150 mM sodium chloride, 0.5% NP-40), then 2 µL RNase inhibitor (M0314, NEB) and 10 µL (1 mg/mL) m6A antibody (Abcam Ab151230) were added, and the sample was incubated on a rotator for 2 h at 4°C. The RNA:antibody sample was transferred to one well of a prechilled 12-well plate and cross-linked twice at 150 mJ/cm2 (254 nm wavelength) using a Stratalinker UV Crosslinker (or equivalent). A total of 50 µL of protein A/G magnetic beads (88803, Pierce) were aliquoted into a fresh tube and washed twice with 500 µL Binding/Low Salt Buffer. The beads were resuspended in 100 µL Binding/Low Salt Buffer, added to the cross-linked RNA:antibody sample, and the bead mixture was incubated at 4°C overnight with rotation. The next day, the beads were washed twice with 900 µL High Salt Wash Buffer (50 mM Tris-HCl pH 7.4, 1 M sodium chloride, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate) and once with 500 µL Wash Buffer (20 mM Tris-HCl pH 7.4, 10 mM magnesium chloride, 0.2% Tween-20). The beads were resuspended in 500 µL Wash Buffer.
4
Crosslinking RNA-Antibody Complexes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Crosslinked RNA-Antibody was generated as previously described [57 (link)]. Fragmented RNA was resuspended in 500 μl binding/low salt buffer (50 mM Tris-HCl (pH 7.4), 150 mM sodium chloride, 0.5% NP-40) containing 2 μl Rnase Inhibitor (M0314, NEB) and 10 μl m6A antibody (ab151230, Abcam), and incubated for 2 hours at room temperature with rotation. RNA-Antibody sample was transferred to 1 well of a 12-well dish and placed in a shallow dish of ice. Sample was crosslinked twice at 150 mJ/cm2 using a Stratagene Stratalinker UV Crosslinker 1800 and transferred to a new tube. A total of 50 μl Protein A/G Magnetic Beads (88803, Pierce) were washed twice with binding/low salt buffer, resuspended in 100 μl binding/low salt buffer, and added to crosslinked RNA-Antibody sample. Beads were incubated at 4°C overnight with rotation.
5
RNA-Antibody Crosslinking and Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Crosslinked RNA-Antibody was generated as previously described (Grozhik, Linder, Olarerin-George, & Jaffrey, 2017) (link). Fragmented RNA was resuspended in 500 µl Binding/Low Salt Buffer (50 mM Tris-HCl pH 7.4, 150 mM Sodium Chloride, 0.5% NP-40) containing 2 µl RNase Inhibitor (M0314, NEB) and 10 µl m6A antibody (ab151230, Abcam), and incubated for 2 hours at room temperature with rotation.
RNA-Antibody sample was transferred to one well of a 12-well dish and placed in a shallow dish of ice.
Sample was crosslinked twice at 150 mJ/cm 2 using a Stratagene Stratalinker UV Crosslinker 1800 and transferred to a new tube. 50 µl Protein A/G Magnetic Beads (88803, Pierce) were washed twice with Binding/Low Salt Buffer, resuspended in 100 µl Binding/Low Salt Buffer, and added to crosslinked RNA-Antibody sample. Beads were incubated at 4°C overnight with rotation.
RNA-Antibody sample was transferred to one well of a 12-well dish and placed in a shallow dish of ice.
Sample was crosslinked twice at 150 mJ/cm 2 using a Stratagene Stratalinker UV Crosslinker 1800 and transferred to a new tube. 50 µl Protein A/G Magnetic Beads (88803, Pierce) were washed twice with Binding/Low Salt Buffer, resuspended in 100 µl Binding/Low Salt Buffer, and added to crosslinked RNA-Antibody sample. Beads were incubated at 4°C overnight with rotation.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!