• 3' Adaptor Ligation and Purification
Prepare 5X T4 RNA Ligase Buffer (no ATP) taken from England et al. PNAS. 1977. 74: 4839.
250 mM Hepes pH 8.3
50 mM MgCl2 Use RNase-free reagents and techniques. Store buffer at –20℃
16.5 mM DTT
50 ug/ml BSA
41.5% glycerol
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 l℃ate 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 ℃cur 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.
Figure 1. 3' Ligation Reaction Timecourse:
