MICROBIOLOGY MIMM 386(Laboratory Course in Microbiology and Immunology)

Dr. Benoit CousineauDepartment of Microbiology & Immunology

McGill University

Exercise 4

BamHIBamHIBamHIBamHIBamHIReclosed vectorClone (good orientation)Clone (wrong orientation)Restriction analysisPCR analysisTransformation(Exercise # 2)(Exercise #4)(Exercise #3)of the good cloneProtein expressionand purification(Exercise #5)Protein gel (PAGE)and immunodetection(Exercise #6)(western blot)

Exercise 4: Preparation of competent cells and transformation of E. coli with plasmid DNA

• Transformation of bacteria:– Introduce a plasmid within the cell (cytoplasm)

• Competent cells:– Cells treated to increase their efficiency for

DNA uptake from the medium– Natural competence is too low– The mechanism is still unknown (empirical data)– Transient state of competence

Different strategies to transform bacterial cells

• Heat shock:– Ice-cold solution of CaCl2 (2-mercaptoethanol)

– 42ºC for 45 seconds (cells + plasmid DNA)– 105-106 colonies/µg of supercoiled plasmid DNA– Plasmid size and form can affect the efficiency

• Electroporation:– Ice-cold water– 2.5kVolts at 4ºC (cells + plasmid DNA)– 109-1010 colonies/µg of supercoiled plasmid DNA– Plasmid size and form can affect the efficiency

Circular vs supercoiled plasmids

LB MgCl2 CaCl2

0

100

200

300

400

500

600

-2ME

+2ME

Conditions

Transformation efficiency (x1000 colonies /ug DNA)

Transformation efficiency in different conditions

T r a n s f o r m a t i o n o f E . c o l i w i t h p l a s m i d D N A

L B

C e n t r i f u g e

P E G C a C l2

D M S O

A d d D N A ( 1 0 - 1 0 0 n g )

H e a t P u l s e E l e c t r o p o r a t i o n

R e c o v e r y a n d c l o n i n g

o n s e l e c t i v e L B p l a t e s

I s o l a t e d c o l o n i e s

E . c o l i

G r o w t o m i d - l o g p h a s e

i n n o n - s e l e c t i v e m e d i u m

DH5 cellsInoculate from a single colony

Grow at 37°COD600 = 0.45-0.55

10µl or less

42ºC for 45 seconds 2.5 kVolts at 4ºC

Add 900 µl of SOC, incubate 1h00 (37ºC),dilute and spread on LB/Amp plates

10864200.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

Time (hours)

O.D. (600 nm)

lag

exponential

plateau

E. coli DH5 growth curve in LB broth