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Gill:Confirming phage via gel electrophoresis: Difference between revisions
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Gill:Confirming phage via gel electrophoresis (view source)
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Open the genomic sequence of your phage in ApE. Go to Enzymes --> Enzyme Selector... (or hit Ctrl + E). In the Enzyme Selection window, go to File --> Open new enzymes file --> Select "REBASE_Available_Enzymes" and click "Open". To simulate a restriction digest, click on an enzyme name (it should become highlighted in red) and then click "Digest". Try out several different enzymes until you find one which gives a reasonable amount of easily distinguishable bands. Use that enzyme to perform the restriction digest. | Open the genomic sequence of your phage in ApE. Go to Enzymes --> Enzyme Selector... (or hit Ctrl + E). In the Enzyme Selection window, go to File --> Open new enzymes file --> Select "REBASE_Available_Enzymes" and click "Open". To simulate a restriction digest, click on an enzyme name (it should become highlighted in red) and then click "Digest". Try out several different enzymes until you find one which gives a reasonable amount of easily distinguishable bands. Use that enzyme to perform the restriction digest. | ||
Label two 1.5 mL Eppendorf tubes-- one for the restriction digest and one for a control without restriction enzyme. Based on the concentration of phage gDNA in your sample, calculate what volume contains 0.5 μg of gDNA. Add the appropriate volume of gDNA, 0.5 μL restriction enzyme, 2 μL NEB Cutsmart buffer, and enough sterile water to bring the reaction volume to 20 μL. For the control, substitute | Label two 1.5 mL Eppendorf tubes-- one for the restriction digest and one for a control without restriction enzyme. Based on the concentration of phage gDNA in your sample, calculate what volume contains 0.5 μg of gDNA. Add the appropriate volume of gDNA, 0.5 μL restriction enzyme, 2 μL NEB Cutsmart buffer, and enough sterile water to bring the reaction volume to 20 μL. For the control, substitute sterile water for the restriction enzyme. Leave both to incubate at 37 °C overnight. | ||
In the morning, run the entire contents of each tube out on a gel (no need to heat-inactivate the enzyme), and visualize the gel. Remember that depending on how the phage replicates its genome, the sequence might not be identical to the actual packaged genomic DNA (e.g. phage K will have long terminal repeats), so some divergence from the predicted banding pattern is expected. | In the morning, run the entire contents of each tube out on a gel (no need to heat-inactivate the enzyme), and visualize the gel. Remember that depending on how the phage replicates its genome, the sequence might not be identical to the actual packaged genomic DNA (e.g. phage K will have long terminal repeats), so some divergence from the predicted banding pattern is expected. | ||
