Bacteriology
L iq u id c u ltu re s w e re g ro w n in L -B ro th , su p p le m e n te d w it h 100 m g / m l a m p ic illin o r 25 m g /m l tetracycline as required. F or agar plates L -B ro th was s u p p le m e n te d w ith 1.5% ( w / v ) bactoagar. B acteria w e re fro z e n in 1 m l a liq u ots o f L -B ro th supplem ented w ith 1 /1 0 v o lu m e o f lOxHogness.
E .c o li T ra n s fo rm a tio n
To p repare com petent cells, a single co lo n y o f E.coli X L l Blue w as p ic k e d fro m agar plates and g ro w n o v e rn ig h t in 10 m l o f L -B ro th at 37®C, 250 rp m in an o rb ita l shaker (N e w B ru n s w ic k M o d e l G25) to s ta tio n a ry phase. Five h u n d re d m l o f L -B ro th was in o cu la te d w ith 5 m l o f the o v e rn ig h t c u ltu re and g ro w n w ith s h a kin g at 37^C u n til an o p tic a l d e n s ity o f 0.5 at 600 n m w as reached. Bacteria w ere then p e lle te d in 50 m l fa lc o n tubes (Falcon, Beckton D ickin so n ) at l,285xg, O^C fo r 20 m in u te s and g e n tly resuspended in 50 m l o f tra n s fo rm a tio n b u ffe r. Cells w ere le ft on ice fo r 10 m in u te s and th e n fro z e n in 400 p i a liq u o ts in s te rile e p p e n d o rf tubes u s in g a d r y ic e /m e th a n o l b a th and stored at -70°C.
F or tra n s fo rm a tio n o f bacteria, a lig a tio n re a c tio n c o n ta in in g 100 n g o f D N A w as m ix e d w it h 20 p i o f 5 x K C M b u ffe r and m ade u p to 100 p i fin a l v o lu m e w it h sterile d d H
2 0
.100
p i o f com petent cells w ere added and le ft on ice fo r 20 m inutes. F o llo w in g in c u b a tio n at RT fo r a fu rth e r 10 m inu tes, 1 m l o f L -B ro th was a dded and cells w ere g ro w n fo r 40 m in u te s at 3 7 ^ Cw ith shaking. 100 g l o f th is c u ltu re was then p la te d o u t o n to L-agar plates su p ple m e nted w it h the a p p ro p ria te a n tib io tic .
P la s m id M in ip re p
Bacteria o f a 10 m l o v e rn ig h t c u ltu re o f L -B ro th su p p le m e n te d w it h the a p p ro p ria te selective a n tib io tic w ere pelleted b y c e n trifu g a tio n at 2,520xg fo r 10 m in u te s at 40C. The bacterial p e lle t was th e n resuspended b y v o rte x in g in 200 p i Ix G lu c o m ix (supplem ented w it h 20 m g / m l R N aseA ), tra n sfe rre d to an e p p e n d o rf tube and incubated at ro o m te m p e ra tu re fo r 5 m in u te s. Bacteria w ere lysed w it h 400 p i o f Lysis M ix and in cu b a te d on ice fo r 5 m inutes. T his was fo llo w e d b y the a d d itio n o f 300 p i 5 M K A c p H 4.8 and gentle m ix in g . D ebris was pelleted fo r 10 m in u te s at 13,000rpm in a bench to p c e n trifu g e and p la s m id D N A w as p re c ip ita te d fro m the s u p e rn a ta n t w ith 0.6 volum es o f iso -p ro p a n o l at RT and collected b y c e n trifu g a tio n in a be nch to p c e n trifu g e at m a x im u m speed. The D N A p e lle t w as th e n resuspended in 100 p i d d H 2 0 and stored at -20^C. Before tre a tm e n t w it h re s tric tio n enzym es, D N A was treated tw ic e w it h th e p h e n o l/c h lo ro fo rm and p re c ip ita te d w it h ethanol.
P la s m id M a x ip re p
Bacteria w ere harvested fro m 1 litre over n ig h t c u ltu re b y c e n trifu g a tio n at 4,550xg fo r 30 m inutes. The p e lle t w as resuspended b y v o rte x in g in 40 m l Ix G lu c o m ix (supplem ented w it h 50 m g / m l RNase A ) and in cu b a te d fo r 5 m in u te s at RT. Bacteria w ere th e n lysed w it h 80 m l o f L y s is M ix fo r 10
m in u te s and c h ro m o s o m a l D N A a n d p ro te in w as p re c ip ita te d fo r 10 m in u te s on ice b y the a d d itio n o f 60 m l 5 M K A c. D ebris was re m o ve d b y c e n trifu g a tio n at 4,550xg fo r 20 m in u te s . The s u p e rn a ta n t w as filte r e d th ro u g h 3 layers o f cheese cloth and p la s m id D N A w as p re cip ita te d at RT b y the a d d in g