National Centre for Biotechnology Education

www.ncbe.reading.ac.uk

Bacterial

Transformation

Copyright © Dean Madden, 2013

Cop

yright ©

Dea

n M

ad

de

n, 20

13

The bacteriumBacterial

chromosome

Plasmids

Ribosomes

Cell wall

Cell membranes

Cytoplasm

Cop

yright ©

Dea

n M

ad

de

n, 20

13

GFP geneThis gene encodes green fluorescent protein, which glows in UV light

Kanamycin resistance genethe enzyme encoded by this gene stops the antibiotic kanamycin from working

Origin of replicationthis is needed so that the plasmid can be copied, using the bacterium’s DNA copying machinery

The plasmid

Cop

yright ©

Dea

n M

ad

de

n, 20

13

Aequorea victoria

Cop

yright ©

Dea

n M

ad

de

n, 20

13

GFP

Cop

yright ©

Dea

n M

ad

de

n, 20

13Glo Fish

Cop

yright ©

Dea

n M

ad

de

n, 20

13

Cop

yright ©

Dea

n M

ad

de

n, 20

13

Cop

yright ©

Dea

n M

ad

de

n, 20

13

Cop

yright ©

Dea

n M

ad

de

n, 20

13

Stock bacteria

These should be grown 1–2 days in advance

Cop

yright ©

Dea

n M

ad

de

n, 20

13

Twiddle vigorously to dislodge the bacteria and

disperse them

Transformation buffer

Cop

yright ©

Dea

n M

ad

de

n, 20

13

Leave on ice for 15 minutes to recover

Cop

yright ©

Dea

n M

ad

de

n, 20

13

Bacterial cell with pores in cell membrane

GFPplasmid

DNA

Cop

yright ©

Dea

n M

ad

de

n, 20

13

LB agar+ kanamycin

+ IPTG

3 drops(100 µL)

Cop

yright ©

Dea

n M

ad

de

n, 20

13

Cop

yright ©

Dea

n M

ad

de

n, 20

13

Label the base of each plate with your initials, the

date and the type of bacteria used

Seal the plates with

tape

Cop

yright ©

Dea

n M

ad

de

n, 20

13

Cop

yright ©

Dea

n M

ad

de

n, 20

13

Cop

yright ©

Dea

n M

ad

de

n, 20

13

Cop

yright ©

Dea

n M

ad

de

n, 20

13

Cop

yright ©

Dea

n M

ad

de

n, 20

13

Cop

yright ©

Dea

n M

ad

de

n, 20

13

BIOHAZARD

TRANSFORMED CELLS MUST BE DESTROYED

AFTER USE