dna science day 2 extracting, ligating and transforming physical biology bootcamp october 2006...
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DNA ScienceDay 2
Extracting, Ligating and Transforming
Physical Biology Bootcamp
October 2006
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 (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 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