Isolation of Plasmid DNA and Analysis of Isolated Plasmid

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 pET11a containing a DNA fragment with the tetracycline resistance cassette. To this end you will be using the Qiagen Mini-Prep kit. (www.qiagen.com). Your instructor has performed some of the steps in the protocol below.

Inoculate a colony representing your cloned DNA into 5.0 mL of liquid media (TCS) containing 100ug/mL ampicillin and 10ug/mL tetracycline. Allow the bacteria to grow overnight at 37oC with vigorous shaking.

Transfer 2.5 mL of the overnight culture into a conical centrifuge tube; spin the cells at 5000 rpm (revolutions per minute) for 5 minutes to pellet the bacteria. Remove the supernatant which is simply the media in which the bacteria was grown.

Resuspend the bacterial pellet in 250 uL of Buffer P1 with RNAse and transfer the resuspended bacteria to a micro-centrifuge tube.

Add 250 uL of Buffer P2 and gently invert the tube 6 times to mix the bacteria and the buffers. The solution should appear somewhat viscous and should become clear. [Buffer P2 contains Sodium Hydroxide (NaOH) and SDS (a detergent) that lysis the bacteria thus releasing the contents of the bacteria. The NaOH/SDS also serves to denature the proteins present in the lysate. The RNase present in Buffer P1 will digest all of the released RNA.]

Add 350 ul of Buffer N3 and IMMEDIATELY invert the tube 6 times to mix the lysed bacteria and the buffers. The solution should become cloudy. Buffer N3 is a high salt buffer (probably contains Sodium Acetate) which causes the relatively large chromosomal DNA and the denatured proteins and cell debris to precipitate into a large insoluble mass while the relatively smaller plasmid DNA remains in solution.

Centrifuge the mixture at 20, 000 rpm for 10 minutes at room temperature. Remove 800 uL of the supernatant that contains the plasmid DNA and place into a clean micro-centrifuge tube. Discard the white pellet which contains the chromosomal DNA, denatured protein and cell debris.

Apply the collected supernatant to a Qiagen column provided by your instructor. Place the column on the manifold also provided by your instructor. Your instructor will apply a vacuum that will allow the supernatant to pass through the column. The buffers will pass through the column into a reservoir while the plasmid DNA will “stick” to the column.

Wash the column containing your plasmid DNA with 600 ul of Buffer PB. Buffer PB contains chaotrophic agents that will remove any excess enzymes that may interfere with the purity of your plasmid DNA.

Wash the column with 750uL of buffer PE.

Place the column into a clean centrifuge tube and spin for 1 minute at 20,000 rpm to remove all of the buffers.

Place the column into a new centrifuge tube and add 50 ul of ddH2O. Allow the water and column to sit for 5 minutes and then centrifuge the column to extract the plasmid DNA from the column. The solution that appears in your centrifuge contains your purified plasmid DNA.

Analysis of the isolated plasmid: To this end you will digest 10 uL of the isolated plasmid with restriction enzyme BamHI. The 40 uL of the plasmid DNA will be quantified using Nanodrop spectro-photometry. A predetermined amount of the DNA will be submitted for sequence analysis.

Remove 10 uL of the plasmid DNA and add this to a micro-centrifuge tube provided by your instructor. This microfuge tube contains 2 uL of buffer for BamHI, 1 uL of the restriction enzyme BamHI and 7 uL of ddH20. The total reaction volume will be 20 uL.

The instructor will incubate the plasmid DNA with the BamHI restriction enzyme for 12+ hours at 37oC then stop the reaction by adding 4 uL of 6X agarose gel loading buffer and by freezing the reactions until the next laboratory period.