Week 5 Wednesday: –Ligation of chromosomal digests into plasmid vectors – part II – Checking ligation success Thursday: –Electrocompetent cell preparation – handout –Test transformation - handout

Ligation of chromosomal digests into plasmid vectors – part II – Checking ligation success Each of the 4 ligation reactions will be compared to the no-ligase samples prepared previously Prepare 80 ml of agarose gel and pour as before Load according to page 87 The disappearance of the vector band in the ligase reactions indicates success

Week 5 Thursday: –Electrocompetent cell preparation – handout –Test transformation - handout

Electroporation and electrocompetency Subjecting E.coli cells to a sharp pulse of electricity results in the formation of transient, transmembrane pores large enough for plasmid DNA to enter To prepare electrocompetent cells: –E.coli cells are grown to mid-log phase –Chill and centrifuge –Wash extensively in ice-cold buffer or water –Resuspend in ice-cold buffer containing 10% glycerol

Electrocompetent cell preparation We will start with a culture inoculated this morning We will follow the protocol in the handout We will step through the procedure as a class It is essential to maintain the cells and all liquids/solutions at 4°C

Test transformation To test the transformation efficiency of the cells, we will transform 5 ng of plasmid DNA (TA ice bucket) Procedure is outlined starting with step 12 The cuvettes must be chilled prior to adding the DNA or the cells

Test Transformation The plasmid DNA must be dialyzed prior to transformation – drop dialysis – DEMO Dialyze 5 μl of plasmid DNA as demonstrated Add 2 μl DNA to the gap; add 40 μl of cells - DEMO Electroporate - DEMO

Test Transformation Immediately after electroporation add 1ml of room temperature LB (change) – mix by inverting the cuvette Incubate for 1 hr at 37°C Transfer the cells to a microfuge tube and spin at 5K for 5 min Resuspend the pellet in 120 μl of LB Create 1:10 and 1:100 dilutions Spread-plate 100 μl of the resuspended cells and both dilutions on LB-Amp media Incubate at 37 °C

Due Wednesday Feb 14: Ex. 9B – Agarose plate fluorescence quantitation of DNA Ex. 10, part I & II – Ligation and ligation check experiments