Gill:Confirming phage via gel electrophoresis
From OpenWetWareJump to navigationJump to search
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 that 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 NanoDrop 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 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.
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- When pipetting restriction enzyme, only touch the tip of the pipette to the surface of the liquid.
- If no gDNA shows up on the gel, nuclease contamination is a possibility. Use a NanoDrop to check for protein contamination and degradation of DNA. If necessary, repeat the phage genomic extraction, making sure not to touch anything with bare hands, changing gloves after steps involving nuclease, and keeping any nuclease-containing waste separate from the phage suspension. Eluting gDNA in TE instead of water will also help prevent degradation by nucleases. When working with a bacterial host that produces a heat-stable nuclease (e.g. S. aureus), be sure to degrade the nuclease using proteinase K during the DNA extraction.
- If gDNA is sticking in the wells, heat in a heat block at 60 °C for an hour before performing the restriction digest to bring it into solution. Storing gDNA at 5 °C instead of -20 °C will also prevent this from occurring.