On a 1% agarose gel, test your PCR product from last week. Mix 8 uL of your PCR product with 2 uL of 5x loading dye. Load the entire 10 uL on the gel. Also add 5 uL of a molecular weigjht marker to each gel. Run the gel at 100 V for 45 minutes.
We will ligate the amplified tetracycline resistance gene into the plasmid pCR2.1. Taq DNA polymerase has the unusual property of adding an extra A to the 5’ end of each strand of DNA that it replicates. To facilitate cloning of PCR products, the pCR2.1 plasmid has been designed to contain a 3’ T on both strands of DNA. Thus, this plasmid has a version of a sticky end that is compatible with the "sticky end" created by the Taq polymerase. By mixing a PCR product with this vector, then, we can generate recombinant molecules. Also, these sticky ends in pCR2.1 occur within the coding region of the lacZ gene. The product of the lacZ gene, b -galactosidase, interacts with X-gal to produce a blue product. When an insert is ligated into this position, though, the lacZ gene is disrupted and b -galactosidase is not produced. As a result, colonies containing a recombinant plasmid will be white, when grown in the presence of X-gal.
In a 0.5 mL tube mix:
1 ul salt solution
1 ul pCR2.1 plasmid
4 ul PCR product
Mix reactions by gently flicking the tube
Incubate at room temperature for 5 minutes
Place tubes on ice
Add 2 uL of ligation mix to tube containing 50 uL of competent E. coli cells
Gently flick tube and place on ice for 30 minutes
Heat-shock cells for 30 seconds at 42oC
Return tubes to ice for 2 minutes
Add 250 uL SOC broth to the tube
Mix tube gently
Incubate at 37oC with shaking for 1 hour
Plate 50 uL of transformation
reaction on LB/amp/X-gal
Plate 50 uL of transformation reaction on LB plate
Incubate plates overnight at 37oC
Observe your plates. Make notes on the number of colonies.
Transfer several colonies to a LB/tet plate
Incubate overnight at 37oC
Observe your plates.