MicroRNA and siRNA Cloning Protocol
Bartel Lab Protocol Updated: July 2005
Prepare 5X T4 RNA Ligase Buffer (no ATP) taken from England et al.
250 mM Hepes pH 8.3 50 mM MgCl2 16.5 mM DTT 50 ug/ml BSA 41.5% glycerol
Use RNase-free reagents and techniques. Store buffer at –20oC
Set a 3' Adaptor Ligation Reaction; Incubate at Room Temp for 2 hrs
2 µL 5x Ligation Buffer 2 µL 100 µM App17.91x 1 µL T4 RNA Ligase (Promega or GE Amersham, FPLC pure) 5 µL purified small RNAs (containing hot labeled RNA markers)
Stop reaction with 15 µL 2X Urea Loading Dye.
Prepare a 10% (0.5 mm) denaturing polyacrylamide gel. Prerun, then load into 2-4 lanes (spread out the reaction to prevent overloading and to dilute the salt in the reaction). Run gel until good separation of BB and XC dyes (about 3-4 inches).
Separate one of the plates, keeping gel on other plate, and cover with Saran wrap. Expose on a phosphor plate, and locate ligated bands (higher mobility- see Figure 1.). Cut out the gel slice that includes the 35'mer and 41'mer ligation product and transfer into siliconized tubes. Avoid the upper and lower ligation artifacts (which occur due to the high ligation efficiency of the adenylated linker). Elute RNAs from gel slice, and ethanol precipitate with glycogen. Resuspend all pellets together into 10 µL dH2O.
Incubation Time (hrs): 0
Upper ligation artifacts
41’mer ligated RNA product 35’mer ligated RNA product
24’mer RNA marker, unligated substrate
18’mer RNA marker, unligated substrate
Lower ligation artifacts
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