Q1: Im using a 1% agarose gel formed in DI water with >1 kb agarose. Is this the correct kind to use for a comet assay to assess DNA damage?
Q2: I've tried running my gels at 30 V 300 mA and 250 W for 30 mins, 60 mins and 120 mins. What settings can you recommend for electrophoresis?
Q3: When I image my gels should I dry them with 80% ethanol and then dry them? Or should I keep them hydrated and mount them with coverslips?
I'm lysing my cells and the negative control is forming nice tight fluorescent balls while the positive control isn't forming any comets at all. I have micrographs of the lysed cells so I know that it isn't to do with that stage. I make my solutions fresh the night before and they are chilled to 4 oC prior to use. All the experiment is carried out in the dark at 4 oC. My positive control has been damaged by 10 Gy on ice to prevent DNA repair. I'm using PI as my stain.
A detailed description of the alkaline Comet Assay protocol is given in the attached document. The possible or common sources of error are also clearly defined. Cheers!
The comet assay protocol need to be modified based on your cell type.Therefore you need to optimized according to your experiment.The basic procedure remains the same,however parameters such as lysis time need to be optimized.I am attaching a document.Hope it will be helpful
I agree you have to standardize the protocol according to cell type and specific goal. It is a general laboratory rule anyhow.
Have you been using LMP agarose?
I remember something about dissolving LMP agarose in a solvent other than water. Let me check it.
For my procedure (based on what I've found online), I'm using a 1% agarose gel in PBS. 0.1g LMP agarose in 10mL PBS. Iman Hassan Ibrahim this may be what you're thinking of?
I've been running my gels for 25 minutes at 25 V, although it could differ depending on what you're running.
Dear Jordon Sandland,
In our work we used a different set up, you can check
Article Klebsiella oxytoca enterotoxins tilimycin and tilivalline ha...
Answers for you
1)Yes
2)You should you use next settings - 300mA (not constant should be adjusted by buffer volume)/ Constant voltage that is equal to diagonal of your electrophoresis chamber in cm. / 30 min
3)Dry with EtOH, and cover with water to store it.during imaging Always cover with water.
Thank you everyone for your help. I am truly grateful. I found that the electrophoresis times were too long. 10-20 mins gave the best results.
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