PreparationofBrothandPlates,etc.

1)LBBroth

Make16gmofLBBrothBase(Gibco#M27800C)upto800mlinddH2O.

Swirltodissolve,thenadd110µlof10NNaOH.Autoclave.

2)NZYBrothCombine:

NaCl4gm(Fisher#S671-500;fromMaterialManag.)

MgSO47H2O1.6gm(Sigma#M-2773)

YeastExtract4gm(Difco#0127-05-3)

N-Z-Amine8gm(Sigma#C-0626)

Makeupto800mlinddH2O.Swirltodissolve,thenadd110µlof10NNaOH.Autoclave.

3)TerrificBrothCombine:

Tryptone9.6gm(Difco#0123-05-7)

YeastExtract19.2gm(Difco#0127-05-3)

Glycerol3.2ml(Sigma#G-5516)

Makeupto720mlinddH2O.Swirltodissolve,thenautoclave20min.

Add80mlof0.17MKPO4(make1.85gmKH2PO4and10gmK2HPO4upto80mlinddH2O;autoclave)andautoclave.

4)LBPlates

Make25.5gmofLBAgar(Gibco#M27000C)upto800mlinddH2O.

Swirl,thenadd110µlof10NNaOH.Autoclave,thenmixwell.

Allowtocoolto50°C(checkwithBioRadtemperatureindicatorstrip)thenadd800µlof50mg/mlampicillinforLB-Amp[red/black],or4mlof10mg/mlkanamycinforLB-Kana[red/red],or1.92mlof5mg/mltetracyclineforLB-Tetra[black/black].

Mixwellbyswirling,thenimmediatelypourontoplatesbyflame.Addjustenoughtocoverbottom.Flamesurfacetoremoveairbubbles.

5)NZYPlatesCombine:

NaCl20gm

MgSO47H2O8gm

YeastExtract20gm

N-Z-Amine40gm

BactoAgar60gm(Difco#0140-01;fromMaterialManag)

Makeupto4000mlinddH2O.Swirl,thenadd900µlof10NNaOH.Autoclavefor75min.Coolto50°C.Pourunderflameonto150mmplates.

6)NZYTopAgarose

Add1.33gmagarose(Pharmacia#170554-02)to190mlNZYbroth.Autoclave.Coolto47°C.Solidified,steriletopagarosecanbemicrowavemelted,thencooledto47°C.

7)Antibiotics

Ampicillinstock,50mg/mlinddH2O,filtersterilize,aliquotandstoreat-20