Abstract: . . .  Monomers, dimmers and trimers 500 700 bp 300 200 100 bp Suggested region of gel slice for purification of concatamers Page 8 MicroRNA and siRNA Cloning Protocol Bartel Lab Protocol Updated: July 2005 Please reference: Lau et al., Science (2001). 294:858-6 Page 8 of 8 • Cloning into TOPO vector  . . .
. . .  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. Figure 6. PCR Screening of Topo Colonies.  . . .
. . .  dimmers and trimers 500 700 bp 300 200 100 bp Suggested region of gel slice for purification of concatamers Page 8 MicroRNA and siRNA Cloning Protocol Bartel Lab Protocol Updated: July 2005 Please reference: Lau et al., Science (2001). 294:858-6 Page 8 of 8 • Cloning into TOPO vector Resuspend concatamers . . .
. . .  dH 2 O 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 . . .
. . .  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. 25 cycles of PCR using the COLONY Protocol 94 o C – 3 min (burst open cells) 94 o C – 30 sec 50 o C – 30 sec 72 o C – 30 sec 3 µL 10X PCR Buffer  . . .
. . .  700 bp 300 200 100 bp Suggested region of gel slice for purification of concatamers Page 8 MicroRNA and siRNA Cloning Protocol Bartel Lab Protocol Updated: July 2005 Please reference: Lau et al., Science (2001). 294:858-6 Page 8 of 8 • Cloning into TOPO vector Resuspend concatamers in the following Taq Fill . . .
. . .  700 bp 300 200 100 bp Suggested region of gel slice for purification of concatamers Page 8 MicroRNA and siRNA Cloning Protocol Bartel Lab Protocol Updated: July 2005 Please reference: Lau et al., Science (2001). 294:858-6 Page 8 of 8 • Cloning into TOPO vector Resuspend concatamers in the following Taq Fill In . . .
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