You can use the gRNA example here as your template.
Although IDT suggests to resuspend at 10ng/µL, higher yields of RNA are obtained at 20ng.
Master mix | 2 | |
Component | Vol | Master mix |
10x Reaction buffer | 2 | 4 |
gBlocks DNA template (20 ng/ul) | 8 | |
ATP (75 mM) | 2 | 4 |
CTP (75 mM) | 2 | 4 |
GTP (75 mM) | 2 | 4 |
UTP (75 mM) | 2 | 4 |
T7 enzyme mix | 2 | 4 |
Total | 20 |
CRITICALRun 1µL mRNA on a standard 2% agarose gel. If you don't see a band, amplify the remaining gBlock DNA fragment with PCR and perform in vitro transcription again.
PAUSEThe mRNA can be stored at -80°C for at least a week.
Add 20mL 100% ethanol to Wash Solution Concentrate.
CRITICALIt's important to perform this step to remove residual wash buffer. If there is residual wash buffer, it might affect the efficiency of your downstream applications.
The size is not precise since the gRNA forms a secondary structure. But the band should still be sharp.
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