DNA Science Day 2 Extracting, Ligating and Transforming APh162 Winter 2005 Caltech

Today’s Plan? Extract the cut vector from an agarose gel Ligate the vector to the lacZ insert Transform cells

Gel Extraction Cut out the band with a razor blade –Shorter wavelengths = more damage to the DNA –Keep it small! What samples should be run next to it as a control? –Uncut plasmid –Ladder! QIAquickSpin.pdf

What Happened to the PCR Product? Digest it using HindIII and KpnI –The single cutter controls are not useful here because we don’t have enough resolution Purify the product using a Qiagen PCR purification kit –100 bp – 10 kbp

Ligation RocheRapidDNALigation.pdf Do different insert:vector molar ratios, total mass < 200ng –3:1 –1:1 –1:3 –No insert control –No vector control Perform a PCR purification afterwards Killer cut? –Get rid of any possible religation

Transformation by Electroporation Stress the cells by putting them in an electric field –They’ll take DNA! –Chemical transformation is also possible (cold=stress) Transform no more than 20 ng of ligation –All the ligations –The original plasmid Estimate the transformation efficiency –Our competent cells are 1:1000 concentrated from OD –1:1E6 dilution and below for non-transformed cells –1:1 dilution and below for transformed cells Show the plates with colonies Create the new Plasmid with Vector NTI

Strains E. coli Genetic Sock: MC4100 –No lacZYA –No lacI, tetR or araC –Peters2003.pdf –Good for our initial transformation, but no inducibility MC4100Z1 –Add lacI, tetR and araC –Lutz1997.pdf