Transformations Protocol

Materials Provided

• LB-AMP plates

• S.O.C media

• 42ºC water bath

• 37ºC shaking water bath

• sterile glass beads

• pUC plasmid

Step 1

Pre-heat an LB-AMP plate at 37ºC for 1 hour

• pre-heating and drying the plate allows media spread onto to soak in faster

Step 2

Thaw an appropriate number of 100µl competent cells aliquots on ice

Step 3

Once the cells have just thawed completely, add DNA (up to 5µl of a standard ligation reaction)

Step 4

Incubate cells on ice for 30 mins

Step 5

Heat shock the cells in 42ºC water bath for exactly 30 secs

Step 6

• IMMEDIATELY incubate on ice for 5 min

Step 7

Add 900µl room temp S. O. C media to each transformation and incubate at 37ºC for 45 min in the shaking water bath

Step 8

• spin transformations at 13 000 rpm for 1 minute to pellet the bacteria

• remove the 900µl of supernatant for disposal

• resuspend the bacteria in the remaining media and pipette onto LB-AMP plate

Step 9

• Add sterile glass beads to each plate and replace the cover

• shake the plate to distribute the cells onto the enitre surface

• dump the beads into the provided beaker when finished

Step 10

Incubate the plates face-down overnight at 37ºC

• the plates and then stored at 4ºC

Theory

Transformation Efficiency

• the relative ability of a bacterial cell preparation to take up external DNA

• in order to compare the ability of one lot of cells to another prepared in a diff way, or at a diff time or place, some standard measure of transformation efficiency had to be established

• the standard is the number of colony forming units (CFU) that are generated from an aliquot of cells in a transformation of 1µg of the pUC19 plasmid

• good transformation efficiency can be up to 1 × 10⁹ CFU/µg of pUC19, but typical numbers are between 1 × 10⁶ - 1 × 10⁸

Counting Colonies in the lab

• since counting 1 × 10⁶ - 1 × 10⁸ colonies is impractical, we transform with a much smaller mass of pUC19 and plate only a fraction of the resulting transformation (e.g. 1/10)

• in this figure, if the plating gave rise to 100 colonies, we would calculate the transformation efficiency to be…

Importance of Testing Transformation Efficency

• if you don’t generate any colonies from transformation with plasmid of interest, determining the transformation efficiency will help establish if the problem lies with your cells/ protocol or with your plasmid

Chemical Competence

• E. coli resuspension in CaCl₂ solution at 0ºC

• under these conditions the Ca²⁺ is thought to create pores in the membrane, assist binding of the DNA to the cell membrane

• the DNA is forced into the cells by applying a 42ºC heat shock, which results in a thermal current that sweeps DNA into the cells

• during the incubation at 37ºC, those bacteria which have survived and taken up the plasmid will be beginning to express the protein required to generate antibiotic resistance (in this case, a β-lactamase enabling them to survive on the selection media)