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I plan on detecting Salmonella on raw meat and after I do the whole Polymerase Chain Reaction (PCR) procedure I plan on loading my samples into a gel using gel electrophoresis and then placing it in a spectrophotometer to determine if there was actual contamination within the meat. If the DNA of Salmonella does show up then what will I need to know about its base pairs? Also about what is the number that I need to be looking for?
Along with this question, I'm not sure what percent gel I should use for the detection of Salmonella. I have it's specific primers, however I'm not quick sure what to do from here or what calculations I need for my gel.
Detecting Salmonella on a Gel
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deleted-297831
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Re: Detecting Salmonella on a Gel
First off--how did you plan to isolate DNA? PCR is very sensitive to contaminating genomic DNA from sources other than the target and if your sample has mostly meat DNA with a little Salmonella DNA then you may get a false signal. Be sure to run a sample of fresh meat that should have no Salmonella as a control.
The size of the amplified DNA depends on the primers that you use. How did you select these primers? If they were purchased commercially then the company can tell you the size of the DNA fragment that you should expect to see on the agarose gel. If the PCR product is only a few 100 base pairs then a 2% gel should work fine: http://www.researchgate.net/post/Which_ ... rophoresis
If you want to be certain that your PCR product is correct you should extract the band from the gel, do a restriction digest with an enzyme that cuts within the Salmonella DNA sequence and run it again on the same kind of gel. This is a fairly technical procedure and you are probably not familiar with it, so you will need some help in the lab if you want to do it. Getting the right size DNA product is usually acceptable without having to do a restriction digest. http://www.researchgate.net/post/Can_I_ ... t_directly
If hope this helps. Post again if you have more questions.
Sybee
The size of the amplified DNA depends on the primers that you use. How did you select these primers? If they were purchased commercially then the company can tell you the size of the DNA fragment that you should expect to see on the agarose gel. If the PCR product is only a few 100 base pairs then a 2% gel should work fine: http://www.researchgate.net/post/Which_ ... rophoresis
If you want to be certain that your PCR product is correct you should extract the band from the gel, do a restriction digest with an enzyme that cuts within the Salmonella DNA sequence and run it again on the same kind of gel. This is a fairly technical procedure and you are probably not familiar with it, so you will need some help in the lab if you want to do it. Getting the right size DNA product is usually acceptable without having to do a restriction digest. http://www.researchgate.net/post/Can_I_ ... t_directly
If hope this helps. Post again if you have more questions.
Sybee
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deleted-297831
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Re: Detecting Salmonella on a Gel
Your feedback really helps! Thank you so much.