Document Actions
Splint Ligation Protocol
Attaching F30P (puromycin) to the end of an mRNA template. Taken Directly from Terry Takahashi's Word Document on this site.
Ligation:
x uL of RNA [y uM] (30uM NAP purified RNA, or 10uM for gel purified RNA)
2.5 uL of F30P [500] (10 uM final concentration)
3.75 uL of splint [500] (15 uM final concentration)
12.5 10X Ligase buffer (NEB)
H2O to 122.5 uL
Add 2.5 uL T4 DNA ligase (NEB)
Incubate at RT for >1 hr
Add 2X 80% formamide loading buffer
Run on urea-PAGE
Run gel until bromophenol blue reaches the bottom of the gel
Excise bands w/ razor or cover slip
Electroelute bands in elutrap
Ethanol ppt eluent
Quantitate, adjust concentration to 5-10 uM