1. DNA Science Day 2 Extracting, Ligating and Transforming Physical Biology Bootcamp October 2006 Caltech

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

3. 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

4. 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

5. 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

6. Transformation by Electroporation Stress the cells by putting them in an electric field –They’ll take DNA! –Chemical transformation is also possible (heat=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 600 0.7 –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

7. Strains E. coli Genetic Sock: http://cgsc.biology.yale.edu/ Explain how we are going to screen. DH5aZ1 –No lacZYA –lacI, tetR and araC