Editing Lambda Red Protocol (section)

== <span style="font-family:Courier,monospace;">Contact</span> ==

*<span style="font-family:Courier,monospace;">[[User:ShawnDouglas|Shawn Douglas]] — posted original protocol, performed once in August 2006.</span>





PCR-product mediated Knockouts a la Datsenko and Wanner (PNAS, 2000)<br/>Charlie Kim<br/>October 2001, modified June 2002, updated June 2004<br/>PCR<br/>Use pKD3 (Cm) or pKD4 (Kan) if you would like to knockout a gene with minimal polarity effects on downstream<br/>genes.<br/>Use pKD13 (Kan), pKD32 (Cm), or pKD81 (Kan from Tn903) to knockout entire operons, single genes, or any<br/>other situation in which polarity shouldn’t matter.<br/>1. Do PCR. I use 50 ul reactions<br/>2. Check PCR product on gel<br/>3. MinElute purify the reaction(s), eluting in 10 ul ddH2O pH 7.0-8.5 (our water is around pH 6)<br/>Alternatively, you can do 1-3 PCR reactions and combine them in a single QIAQuick PCR cleanup. Speedvac<br/>to concentrate if necessary/desired. Lately, I’ve just been doing just 1 PCR reaction and doing a standard<br/>Qiaquick. I electroporate 2-5 ul of this, and it works fine.<br/>Electroporation

Because this technique demands electroporation and linear DNA recombination in a single step, the yield of colonies<br/>is generally low. I often see 1-2 colonies from my transformations, but I’ve also recovered as many as 150. My<br/>belief is that one of the most important things is to get the competent cells as dense as you can without causing the<br/>electroporation to arc.<br/>1. Grow cells 5-50 ml 2XYT + 100 ug/ml Amp LT2/pKD46 (I’ve also successfully made knockouts in<br/>TR7065/pKD46 (aka LB5010/pKD46) and 14028S/pKD46, but these strains are not as good for P22<br/>production) – 4-6 hours 30 - 32 degrees C. 10 ml is generally sufficient for doing one knockout.<br/>2. Induce the Red recombinase with 10 mM L-arabinose for 1 hour (inducing for the entire time works fine,<br/>too).<br/>3. Harvest in small rotor floor centrifuge, 10 min 8k rpm<br/>4. Resuspend pellet in 1 ml cold ddH2O (I find that my transformations work fine without any glycerol), spin<br/>30 sec in high-speed Eppendorf microfuge (14k rpm in Eppendorf 5417C)<br/>5. Wash 4 more times as in step 4. (Note: I do these quick washes for speed and convenience; you can<br/>certainly use a standard washing protocol in a big centrifuge, but this way, I can get all of my washes done<br/>in 5 minutes).<br/>6. Resuspend in 40-250 ul cold ddH2O. Generally, I find that about 1.5X – 2X the pellet volume results in<br/>good density (high, but not enough to cause arcing with cells alone).<br/>7. Electroporate (I use 2 mm gap cuvettes, 2.5 kV), recover at 37 degrees C in 1 ml 2XYT. I recover in the<br/>cuvette without agitation for convenience. Recovery up to 3 hours may be worthwhile, but after that I’ve<br/>never seen any increase in yield of clones.<br/>8. Plate on LB with antibiotic, incubate at 37 degrees C<br/>Additional Notes<br/>1) While 1 mM L-arabinose seems to work fine for E. coli, 10 mM seems to work better for Salmonella in my<br/>hands.<br/>2) It is important to titrate your drug concentrations. I had low efficiencies making Cm(R) strains, but I now<br/>know that it was because I was simply using too high a concentration. I went from 30 ug/ml down to 8<br/>ug/ml and the efficiency is similar to making Kan(R) knockouts.<br/>3) I don’t think that the length of arabinose induction is critical, because I’ve obtained mutants inducing for<br/>the entire growth of the culture or for just 1 h before harvesting. I tend to do it for just 1 h before<br/>harvesting because I have the vague notion that I get more recombinants this way, but I’ve never tested this<br/>rigorously.&nbsp;