Extraction of Plasmid Dna
Isolation of plasmid DNA and analysis of isolated plasmid
Introduction: A plasmid is an autonomously replicating extra-chromosomal genetic element. In other words, this is a DNA molecule external to the bacterial chromosome that is able to replicate on its own and distribute its daughter molecules to daughter cells. You have successfully cloned a fragment of chromosomal DNA containing a tetracycline resistance cassette into a plasmid (pET11a). To this end you have (1) isolated total chromosomal DNA from your assigned bacterial strain and digested this DNA with the restriction enzyme BamHI. You have also (2) digested the plasmid cloning vector (pET11a) with the same restriction enzyme. You have (3) randomly ligated the digested chromosomal DNA into the plasmid and (4) transformed an Escherichia coli strain JM109. You have selected for transformants by plating the transformed DNA onto rich media (TCS) containing ampicillin. (The pET11a plasmid contains an ampicillin resistance cassette that renders any bacteria harboring this plasmid resistant to ampicillin and therefore capable of growing on media containing ampicillin). Finally, you have (5) scored for transformants that are not only resistant to ampicillin but also resistant to tetracycline. To this end you replica plated the colonies that grew on the TCS-ampicillin plates onto TCS media containing both ampicillin and tetracycline. If a colony grew on the TCS ampicillin, tetracycline plate then that colony is resistant to ampicillin because the bacteria in that colony contain the pET11a plasmid and is resistant to tetracycline because you have cloned a fragment of DNA that contains the tetracycline resistance cassette. You are now ready to isolate the plasmid containing your cloned fragment such that you can 1. digest the plasmid with BamHI to evaluate the size (bp) of your cloned fragment and 2. submit the plasmid containing your cloned fragment for sequence analysis.
Isolation of the plasmid
Introduction: A plasmid is an autonomously replicating extra-chromosomal genetic element. In other words, this is a DNA molecule external to the bacterial chromosome that is able to replicate on its own and distribute its daughter molecules to daughter cells. You have successfully cloned a fragment of chromosomal DNA containing a tetracycline resistance cassette into a plasmid (pET11a). To this end you have (1) isolated total chromosomal DNA from your assigned bacterial strain and digested this DNA with the restriction enzyme BamHI. You have also (2) digested the plasmid cloning vector (pET11a) with the same restriction enzyme. You have (3) randomly ligated the digested chromosomal DNA into the plasmid and (4) transformed an Escherichia coli strain JM109. You have selected for transformants by plating the transformed DNA onto rich media (TCS) containing ampicillin. (The pET11a plasmid contains an ampicillin resistance cassette that renders any bacteria harboring this plasmid resistant to ampicillin and therefore capable of growing on media containing ampicillin). Finally, you have (5) scored for transformants that are not only resistant to ampicillin but also resistant to tetracycline. To this end you replica plated the colonies that grew on the TCS-ampicillin plates onto TCS media containing both ampicillin and tetracycline. If a colony grew on the TCS ampicillin, tetracycline plate then that colony is resistant to ampicillin because the bacteria in that colony contain the pET11a plasmid and is resistant to tetracycline because you have cloned a fragment of DNA that contains the tetracycline resistance cassette. You are now ready to isolate the plasmid containing your cloned fragment such that you can 1. digest the plasmid with BamHI to evaluate the size (bp) of your cloned fragment and 2. submit the plasmid containing your cloned fragment for sequence analysis.
Isolation of the plasmid