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MicroRNA and siRNA Cloning Protocol

Bartel Lab Protocol Updated: July 2005

Resuspend concatamers in the following Taq Fill In Reaction. Incubate at 72oC for 5 min.

    • 11.5

      µL dH2O

    • 1.5

      µL 10x PCR Buffer

    • 1.5

      µL dNTPs

    • 0.5

      µL Taq polymerase

Have the TOPO TA cloning kit reaction tube set up. Use 5 µL from the fill in reaction for a TOPO-TA Cloning reaction, and freeze the remaining fill-in reaction for storage. Use all of Topo reaction for transformation into chemical competent cells, add 500 µl SOC media, and let the cells grow for only 45 min (not longer) before plating out 50 µL , 150 µL, and 300 µL of the culture on to LB Amp S-Gal plates. Grow overnight at 37oC.

Pick white colonies, and restreak on a master plate. Let this master plate grow ON. Screen only white colonies by PCR in a 96-well microplate format – 30 µL reactions per well.

3 µL 10X PCR Buffer 3 µL 10X dNTPs

    • 0.2

      µL 100 µM M13F

    • 0.2

      µL 100 µM M13R

    • 0.5

      µL Taq Polymerase

23 µL dH2O

25 cycles of PCR using the COLONY Protocol

9 4 o C 3 m i n ( b u r s t o p e n c e l l s ) 9 4 o C 3 0 s e c 5 0 o C 3 0 s e c 7 2 o C 3 0 s e c

Pick colonies from master plates with a pipette tip, swish around in a PCR reaction well. Check completed reactions on a 2% agarose gel, and look for inserts greater than 220 bp (expect 500- 800 bp inserts, see Figure 6). You can now either purify remaining PCRs and sequence directly, or regrow colonies to extract plasmids. Submit to commercial sequencing facility, using M13F or M13R as sequencing primers.

Positive colonies containing concatamers of small RNA clones

– ladder

Product from empty Topo vector

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