Colony PCRs were performed on each of the three transformations from yesterday (16s, EHR, and EUB primers) using the M13F/R vector primers. Colonies were picked form the transformation plates with pipette tips, re-streaked on a secondary, gridded, numbered LBAmp50+x-gal plate and then used to inoculate the respective PCR reactions. Six white colonies (positive clones) and a single blue colony (negative clone) were selected from each transformation.
Restreaked plates were incubated @ 37C O/N and then stored @ 4C (Parafilmed).
Master mix calcs are here: 20150227 – Colony PCR Clam RLO
30μL of each reaction was run on a 1% agarose 1x Low TAE gel, stained w/EtBr.
Ladder: Hyperladder I (Bioline)
Upper Left: 16s colonies 1 – 7
Upper Right: EHR colonies 1 – 6
Lower Left: EUB colonies 1 – 7
Based on the PCRs used for cloning, all white colonies screened exhibit the expected product sizes. Additionally, each of the blue (negative) colonies, produced the expected band size that are indicative of an empty plasmid.
Will select a positive colony from each set for mini prep and Sanger sequencing.