Week 5

1
Week 5

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

2
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

3
Week 5

• Thursday
• Electrocompetent cell preparation handout
• Test transformation - handout

4
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

5
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 4C

6
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

7
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

8
Test Transformation

• Immediately after electroporation add 1ml of
  room temperature LB (change) mix by inverting
  the cuvette
• Incubate for 1 hr at 37C
• Transfer the cells to a microfuge tube and spin
  at 5K for 5 min
• Resuspend the pellet in 120 µl of LB
• Create 110 and 1100 dilutions
• Spread-plate 100 µl of the resuspended cells and
  both dilutions on LB-Amp media
• Incubate at 37 C

9
Due Wednesday Feb 14

• Ex. 9B Agarose plate fluorescence quantitation
  of DNA
• Ex. 10, part I II Ligation and ligation check
  experiments