DNA Science Day 2 Extracting, Ligating and Transforming

1. DNA ScienceDay 2Extracting, Ligating and Transforming APh162 Winter 2005 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 (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 OD600 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/ • 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