The usefulness of PCR extends beyond replicating a target segment. Unfortunately, PCR cloning can be difficult due to the 3’ adenosine bases added to the ends of PCR products by Taq polymerase. This experiment details cloning a gene from S. cerevisiae such that it is compatible with the TOPO TA cloning process. These adenosine overhangs can be used in TOPO TA cloning to easily ligate the PCR products into a specialized vector that is ready for transformation in competent cells.
PCR 1. Label 2 PCR tubes as 50 ng and 100 ng yeast genome DNA.
Label 2 PCR tubes as no DNA control and no primer control.
Add 2 ul primer and 2ul dNTP to each tube.
Add 50 or 100 ng of the appropriate plasmid to the appropriate tubes.
For each tube, add 5 ul 10X reaction buffer and 0.25 ul of Hotstart Taq DNA Polymerase, and ddH2O to bring the total volume to 50 ul.
Place PCR tubes in thermocycler and use the following settings:
94 C for 1 minute
94 C for 1 minute, 62 oC for 1 minute, 72 C for 2 minutes (30 cycles)
72 C for 5 minutes
Hold at 10 C
TOPO Cloning 1. Combine the cloning reagents as shown in table 1. Ideally include one reaction with only the pCR 2.1 vector as a control.