Question

1) The pGLO plasmid does not have EcoR1 sites flanking the GFP/araC region. Why is this an issue and how would it be dealt with?

2) Explain why it was necessary to "clean up" PCR products before performing restriction digestion. Predict what would have happened if we skipped this step.

Answer 1

1. If the pGLO plasmid does not have EcoR1 sites flanking the GFP/araC region the bacteria won’t be glowing under the UV light. It also help to control production o mRNA, and the corresponding protein by the cloned gene. Transcription factors help the cells to turn transcription “on” or “off”. The transcription of the gene in pGLO is controlled by using a araC which works only in the presence of arabinose. The AraC protein, encoded by the araC gene on the pGLO plasmid, is the transcription factor necessary for this control. The protein encoded by araC gene binds to arabinose, which help production of GFP protein and the bacteria will produce fluorescence.
2. Clean-up help to remove single-stranded or double-sranded PCR amplification products (100 bp to 10 kb), excess primers, nucleotides, DNA polymerase, oil and salts etc. After PCR reaction all these components impurities which will interfere with subsequent manipulations such as DNA sequencing or restriction digests. If you skip this step, you won’t get an accurate results with proper bands in the gel. Some unwanted bands will be present in your gel.