• 5' Adaptor Ligation and Purification
Set a 5' Adaptor Ligation Reaction; Incubate at Room Temp for 6 hrs
2 μL 5x Ligation Buffer
2 μL 200 μM 17.93R
1 μL 4 mM ATP
1 μL T4 RNA Ligase
5 μL small RNAs from 3’ Adaptor Ligation Reaction
Stop reaction with 10 μL 2X Urea Loading Dye. Prepare gel and purify 5' adaptor ligation products in the same way as for the 3' ligation products. For band identification, use freshly kinased 10bp ladder as a reference for size. Cut out the 52-60'mer products, and leave behind the unligated 35-43'mers (see Figure 2). Resuspend pellets in a total 10 μL dH2O.
Figure 2. 5’ Ligation Reaction Timecourse:
• RT-PCR of small RNAs with Adaptors
Using siliconized tubes, set up a reverse transcription reaction:
Incubate reverse transcription reaction at 48℃ for 1 hour. Next, add 1 μL RNase H and incubate at 37℃ for 30 minutes. Do all steps in parallel with the (-)RT control. Remaining RT reaction may be stored long term at -20℃.
Set up 100 μL reactions for the RT(+) and RT(-) samples for PCR.
