DNA ScienceDay 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 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

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