Difference between revisions of "Gill:Confirming phage via gel electrophoresis"
(New page: A simple way to confirm the identity of a phage is to extract its genomic DNA, perform a restriction digest, and check if the banding pattern matches what is predicted based on its known s...)
Revision as of 09:44, 25 June 2013
A simple way to confirm the identity of a phage is to extract its genomic DNA, perform a restriction digest, and check if the banding pattern matches what is predicted based on its known sequence.
- 2 1.5 mL Eppendorf tubes
- 0.5 μg phage genomic DNA x 2
- 0.5 μL restriction enzyme
- 2 μL NEB Cutsmart buffer x 2
- Sterile MilliQ H2O
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 which 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 more 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 available 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.
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!