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Sometimes I can not get the house-keeping geen from negative samples. How can I improve?

First, you should check if the standards performed normally. If you could not get any bands from standards, that indicates the PCR failed. You should check the PCR procedure step by step. If the standards performed OK, then the problem should come from the bad DNA quality.

Second, you should check if you used the proper DNA extraction method. The Lysis Buffer is only good for the pleopods, muscle, and shrimps younger than PL 15. If you used Lysis Buffer to extract other samples such as PL 20 or hepatopancrea, you would not be able to get the house-keeping gene.

Third, if the DNA extraction method was qualified, you should focus on the DNA concentration and DNA/protein ratio. You could check OD260 and OD260/280 by spectrophotameter. Or you could dilute the DNA sample by ddH2O 5 times and run PCR again. If you could get the house-keeping gene after dilution which means the original DNA or protein concentration was too high. If you still could not get the house-keeping gene after dilution, the DNA concentration might be too low, and you could use less ddH2O to resuspend the DNA pellet.

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Why did I get the smear pattern from the gel electrophoresis?

Too much DNA, protein, and too strong non-specific reaction will cause the smear pattern. Too strong background after EtBr staining can increase the smear. Actually, slight smear pattern is normal for PCR, especially for the multiplex system. To avoid smear problem, you can adjust the DNA concentration in the beginning. Then, increase the destaining time to wash out the background, or use a thinner gel to run electrophoresis. If the smear was still very strong, even affecting the data assay after the above modification, which means smear should come from the non-specific reactions. We should focus on the samples and check where the non-specific reaction came from.

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Why did I get the non-specific bands?

Non-specific bands usually came from the house-keeping gene primer set. Because we designed the house-keeping gene from a very conserve gene fraction of shrimp, most crustaceous animals also have that gene with high homology. But the size might be different. PCR is a very sensitive method, even very few templates can be amplified successfully. For example, when we check the PL samples, if the animals have eaten other smaller crustaceans, their DNA would be co-extracted. And we might get a different-size house-keeping gene from these samples.

However, the product sizes of those non-specific bands are different from the specific bands, they will not influence the data assay.

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How do I avoid the PCR contamination problem?

The contamination may come from the final PCR products or cross contamination during extraction and reagent-adding step. To avoid the contamination from the PCR products, the best way is to isolate the gel electrophoresis room far away from the sample preparation and reagent preparation room. About the cross contamination problem, a good training is the best way to prevent it.

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