Editing Confusion Cloning (section)

== Home made extract ==

<br>1, Grow DH10B or Top10 with pBad24-Red-alpha at 37 oC in 100 ml 2X YT medium until they reached OD600 ~ 5.3.<br>2. Add 0.2% L-arabinose to induce for 2 h to express lamda prophage protein Red-alpha.<br>3. Harvest cells by centrifugation at 4 oC.<br>4. Wash cells 1 time with 200 ml ddH2O and resuspend cells in 1.2 ml CelLytic B Cell Lysis Reagent (Sigma) or add lysozyme.<br>5. Incubate the resuspended cells at room temperature for 10 minutes to allow lysis to occur.<br>6. Spin cell lysates at 12000 rpm for 10 min at 4 oC to pellet the insoluble material. Collect the resulting supernatant.<br>7. Mix cell extracts with equal volume of 100% glycerol,<br>8. Aliquote into 40–60 ul per tube, store at -20o C or -80 oC for long-term storage