Transformant selection on bacterial lawns using the V18-Tn5-cassette

Contributed by Harry MacWilliams, January 2000, published in Wetterauer et al 1996.
Download word file.

---

[MATERIALS] - [INDEX]

Procedure

Note: Directions are for transforming one vector; increase numbers of plates appropriately for more.

Day 1:

Transfect as you usually do, and make make two plates, each with ca 2x10⁷ cells. Prepare G-100 plates and Ka plates.

Note: I use the calcium phosphate method and 20 µg DNA per plate, and shock with 16% glycerol. I dont see why other transfection methods should not work as well.

Day 2:

Harvest cells from one of the trafo plates. Spin down and suspend in KK2 at about 5x10⁷/ml. Pipette 500 µl Ka suspension and 100 µl cells (about 5x10⁶ cells) onto each G100 plate, spread uniformly, dry in hood. The lawn will look almost transparent when dry (if you read it in the spectrophotometer at 540 nM, blanking on an area of the plate without a lawn, it will read 0.1 to 0.2).

Note: It is perfectly possible to get a thicker lawn with more bacteria, but DONT DO IT as the selection will not work as well.) The second trafo plate is just to allow you to compare bacterial and liquid selection. It gets G20 for two days, after which the medium is exchanged for G8. When clones appear, go back to G20. In liquid, V18Tn5 behaves about the same as A6tn5.

Days 3-7:

Incubate the bacterial selection plates at 22°C.

Note: Depending on the vector, you will get something like 3-100 clones per plate. Macroscopically visible clones are visible on day 3-4; aggregation begins on day 5-6. Transformants develop normally through fruiting on the G100 plates. When you see clones containing the stages that interest you, test for reporter expression as appropriate. In the case of GFP, you can see observe the trafo plates directly (they have much lower background fluorescence than SM plates). For luc, cut out a section of clone, suspend in buffer, pellet cells, freeze, and test. For gal and /or gus, make colony lifts from the selection plates, fix and and stain. If you make lifts, it is a good idea to wait until spores are present in your clones, as the nitrocellulose sometimes removes all of the vegetative cells. Some spores always remain on the agar and can recovered later. After having identified clones with good expression, you can reclone on the same G100 plates (with us pregrown bacteria) as used for selection. The cloning efficiency of transformants is near 100%. To start an axenic culture, amplify your clone on 1-2 G100 plates. When the plates clear (about two days), suspend the cells in buffer, wash once or twice, then resuspend in HL5/G20/penstrep.

[TOP] [INDEX]

---

Materials

• KK2 agar plates
  • 1.5% agar in 1x KK2, autoclaved.
  • 
    
  Note: These should be poured LEVEL, ie, they should be allowed to cool on a precisely horizontal surface; if stacked while cooling, check to see that the stacks are really straight. The plates should not be wrapped up immediately after hardening, but allowed to dry overnight. I just put the stacks of plates, closed of course, on the bench; the laminar flow hood is also OK, but it should be turned off so the plates do not dry out on one side. It is also possible to make these plates further in advance; they are stable for months in the refrigerator.
• 
  
• Ka plates
  Spread an SM plate with Klebsiella aerogenes and put at 37°C overnight. These can also be made in advance and stored (at 4°C they are good for at least a month).
• 
  
• Ka suspension
  Suspend the Ka from the SM plate in 2.5 ml KK2, add penicillin to 100 units/ml, strep to 100 µg/ml and G418 to 100 µg/ml.

• [TOP] [INDEX]