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MicroRNA and siRNA Cloning Protocol

Bartel Lab Protocol Updated: July 2005

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.

Incubation Time (hrs):

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58’mer ligated RNA product 52’mer ligated RNA product

41’mer unligated RNA substrate

35’mer unligated RNA substrate

Using siliconized tubes, set up a reverse transcription reaction:

5 µL of ligated RNAs 1 µL 100 µM 15.22 10 µL dH2O

Heat to 80oC for 2 min Spin down to cool

6 µL 5X First Strand Buffer (Invitrogen) 7 µL 10X dNTP’s 3 µL 100mM DTT 1 µL SuperScript III RT (200U/µL) final

Heat to 48oC for 2 min before adding RT. Take out 3 µL for a (-)RT control.

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