Datsenko-Wanner

Small Scale Electrocompetent Prep and Transformation of pKD46 for Datsenko-Wanner

1. Grow o/n of pKD46 transformed cells at 30oC in LB/Amp.
2. Inoculate 5mL 2YT + amp with 200µL of O/N.
3. Grow uninterrupted for 3 hours uninduced (for MC1061) at 30oC.
4. Add arabinose to 0.2% (100x with 20% stock), let grow additional 1 hr
   1. When you induce, it is a good idea to put your 10% glycerol into the freezer to chill it
5. Put flask on ice; transfer 1.5 mL to epi tube.
   1. For the following discard supe steps, it is extremely important to discard as much supernatant as possible. Flick 3 times or use a pipette
6. Spin down for 30 sec at 14.1 rpm, discard supe, place immediately on ice.
   1. Let sit, discard supe again.
7. Resuspend with .5 mL 10% ice cold glycerol.
8. Spin down for 30 sec (14.1 rpm), discard supe, place immediately on ice.
   1. Let sit, discard supe again.
9. Resuspend with .5 mL 10% ice cold glycerol.
10. Spin down for 30 sec (14.1 rpm), discard supe, place immediately on ice.
    1. Let sit, use pipet to discard supernatant
11. Resuspend in a small volume (50-100 ul) 10% ice cold glycerol.
12. Add ~2.5uL DNA to cells, pipette & stir to mix.
    1. avoid bubbles
13. Immediately add to chilled electroporation cuvette.
    1. Pipette down the side to avoid bubbles
14. Immediately shock
    1. hit the button ONLY TWICE. if you hit it a third time, it will prematurely discharge
    2. make sure time constant is greater than 4; if you hear a pop or if the time constant is low then arcing has occurred and all your cells are dead
       1. Josh: in practice 5.4 is the gold standard. If 4 < timeconstant <

5.4, then your cells are probably dirty.

1. Immediately add 250 uL 2YT, pipette up and down to recover cells, move to tube and keep warm (yay ears!)
   1. this is the most important "immediately" step
2. Put in shaking 37oC incubator for 1 hr
3. Plate everything on appropriate antibiotic plate (allow to dry before putting in 37oC incubator)
   1. Josh's most common sources of failure:
      1. No colonies due to insufficient DNA. Make sure your PCR is a hearty one!
      2. Dead bugs due to bad comp cell prep. Make sure to wash away the salts!
      3. pKD46 not clearing. In this case, pick 10 colonies and spot on Kan and Amp. For the one with the weakest AmpR phenotype, streak out from the Kan plate and pick 10 colonies to spot on Kan and Amp again.

Small Scale Electrocompetent Prep - Generic

1. Grow up strain to saturation
2. Innoculate 2YT with 1:30 of o/n.
3. Grow uninterrupted for 2.5 hours @ 37oC.
   1. Josh: use 4hrs @ 30C for ubercompetency
4. Cool tube/flask; transfer 1.5 mL to chilled epi tube.
5. Spin down for 20 sec at 14.1 rpm, discard supe, place immediately on ice.
6. Resuspend with .5 mL 10% ice cold glycerol.
7. Spin down for 30 sec (14.1 rpm), discard supe, place immediately on ice.
8. Resuspend with .5 mL 10% ice cold glycerol.
9. Spin down for 30 sec (14.1 rpm), discard supe, place immediately on ice.
10. Resuspend in a small volume (90 ul) 10% ice cold glycerol.
11. Add ~2uL DNA to cells, pipette & stir to mix.
12. Immediately add to chilled electroporation cuvette.
13. Immediately shock
14. Immediately add 200 uL 2YT, pipette up and down to recover cells, move to tube and keep warm
15. Put in shaking 37oC incubator for 1 hr
16. Plate everything on appropriate antibiotic plate (allow to dry before putting in 37oC incubator)