Dear Jamal

Firstly, you must detect the purity and concentration of DNA before doing PCR amplification and agarose gel electrophoresis by calculating optical density at 260 and 280 nm and detect the yield. If the yield is less than One Two, it means contamination of DNA with protein and if it's more than Two, it refers to contamination with phenol. In both cases, re-extraction is recommended and achieve agarose gel electrophoresis secondly. Also, duplicating of PCR program amplification is recommended to ensure all PCR reagent mixture contents going on just a target DNA piece.

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Dear Jamal

Firstly, you must detect the purity and concentration of DNA before doing PCR amplification and agarose gel electrophoresis by calculating optical density at 260 and 280 nm and detect the yield. If the yield is less than One Two, it means contamination of DNA with protein and if it's more than Two, it refers to contamination with phenol. In both cases, re-extraction is recommended and achieve agarose gel electrophoresis secondly. Also, duplicating of PCR program amplification is recommended to ensure all PCR reagent mixture contents going on just a target DNA piece.

Hope to benefit from the answer

are you growing the colonies first then making dna or just picking colonies off the plate and doing a dna preparation?

HI Jamal,

if you're using agarose gels to detect and see DNA integrity, I guess you don't have other possibility to do else. probably you need more DNA to be detected on agarose gels. it is said that DNA bands appear at 50ng on agarose gels. when extracting DNA, can you see the DNA bullet? if not, I'm not sure you extracted something.

fred

• Agreed
• Check your concentration and purity by Nanodrop or spec but keep in mind that absorbance @ 260nM can also be linked to phenol and trizol or other aromatic compounds as well as certain complex salts
• In consequence absorbance @ 260nM can over estimate the amount of DNA in impure samples that might have impurities augmnenting the 260nM absorbance
• Also keep in mind that DNA below 20ng will not really show up on a stained agarose gel
• In addition, if your extraction is less than optimal you might end up losing DNA such that what you run on a gel is undetectable, if hat you load is actually < 20ng
• Do your samples that produce visible staining look smeared suggesting sub optimal extraction and/or recovery
• It might be as well to re purify
• Do you have a pic of agarose gel you could provide ?>

If you didnt detect the DNA band, this is not necessary mean there is no DNA! May be the DNA mount is less than detectable. In this case, i prefer to do a universal amplification like ITS.

I agree with Ayoob

Thank you all for your suggestions and answers. I repeated the experiments where I did DNA Extraction by using DNA kit and I got band when I checked the DNA concentration by gel electrophoresis. Then, I did PCR analysis and I got band for all samples.

Instead of I picked a colony from the plate and did DNA extraction, I picked a colony and grown in a 5 ml broth and then did the DNA extraction.