Dorman:Chromosomal knock-outs with tetRA

I've successfully used λ-red recombination to move PCR products containing the tetRA element to knock out genes in E. coli. The protocol below is modified from Karlinsey #Karlinsey-2007.

What you'll need:
- DNA from a strain with a Tn10 element

- Phusion PCR kit. (You can use another polymerase, but you'll need to modify the PCR conditions.)

- PCR primers to amplify tetRA

- PCR primers to verify the insertion

Designing PCR primers
Primer tetR: 5' 40 bases of homology to the chromosome (on sense strand) + TTA AGA CCC ACT TTC ACA TT 3'

Primer tetA: 5' 40 bases of homology to the chromosome (on nonsense strand) + CTA AGC ACT TGT CTC CTG 3'

Karlinsey notes that if you are removing a coding gene, its a good idea to leave the stop codon.

Amplifying the element
PCR reaction:

DNA: 0.2μL 5x HF buffer: 20 dNTPs: 2 tetR: 2 tetA: 2 phusion: 1 H2O: 72.8

Cycling conditions:

98°C: 30s

30 cycles of:

98°C 5s 64°C 15s 72°C 1min, 30s

finishing with:

72°C 5 min

Verifying and purify PCR product
-Run out 4μL of PCR product on an agarose gel to confirm that you've got a 1990 bp fragment

-Purify the PCR product with a PCR cleanup kit, and elute in 30 μL of elution buffer. (Remember to preheat the elution buffer, as recommended by the manufacturer.)

- Check the concentration of your DNA on the nanodrop. You're going to want to add between 100 to 200 ng of DNA, so as long as you can do this in a few μL, you can go ahead to the recombination step. Otherwise, you'll need to concentrate your sample first.