Question

When we isolated plasmid in the second week of this experiment from XL1-blue, we added EDTA to the solution in order to chelate Mg²⁺ ions. Why?

Then when we performed primer extension in vitro, we added Mg²⁺ to the solution. Why the difference?

Answer 1

1. TE buffer for DNA storage contains EDTA which is inhibiting DNases (since it's chelating Ca2, Mg2) and therefor protects the DNA from degradation. In case you have some important DNA samples you should go for TE buffer. If you have samples from cells etc. that are easily reproducible or you don't need them for longer time, you can just use water.

2. Reverse transcriptases (RTs) are RNA-directed DNA polymerases that were first identified as part of the retroviral life cycle. RTs catalyze the synthesis of a DNA copy (cDNA) of the target RNA molecules using a reverse transcription primer, dNTPs, and Mg2+ or Mn2+ as a cofactor. Reverse transcriptases have been adapted for use in a variety of in vitro applications including real-time and endpoint RT-PCR, labeled-cDNA probe generation and cDNA library construction. The ideal reverse transcriptase is robust (highly active under a variety of conditions) and converts all primed RNA within a sample to cDNA, regardless of its abundance, length or secondary structure.