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What is the difference between the Lysis buffer and DTAB/CTAB solution?

DTAB and CTAB are strong cationic detergents. In the DTAB/CTAB extraction procedure, an organic solvent extraction step is included to remove protein. Consequently, the DNA recovery rate and extracted DNA quality are better than that of Lysis Buffer method. The DTAB/CTAB method is suitable for the "dirty" samples which contain more protein or PCR inhibitors, such as hepatopancrea, eye ball, and pond dirt.

Lysis Buffer is an easy-operating method. It is suitable for routine, large sample size operation. Because it is a simplified method, its DNA quality is not as good as that of DTAB/CTAB. Therefore, it is only useful for extracting pleopod (swimming legs), muscles, or seeds below PL15.  

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Should I grind the shrimp tissues very hard to recover more DNA?

This will depend on the sample condition.  It is easier to recover DNA from the frozen samples but more difficult from the ethanol preserved samples. When fresh or frozen samples were used, please do not grind too hard while extracting; otherwise, you will recover too much DNA and protein. You can check the DNA pellet to see if this problem exists. You will get a big, orange DNA pellet if the grinding procedure is too hard. Usually, you only need to push the muscle out of the shell when fresh or frozen samples were used.

On the contrary if you process ethanol fixed samples, you have to grind harder to recover enough DNA. Because DNA in ethanol fixed sample is more difficult to be released as a result of "harder cell" caused by ethanol denaturaling. The DNA pellet obtained from ethanol fixed sample is usually smaller; therefore, a reduced volume of ddH2O or TE buffer to dissolve DNA pellet is recommended.

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Which shrimp organ may contain PCR inhibitors?

Eye balls are known to contain PCR inhibitors. When you extract DNA from eye stalks by Lysis Buffer, eye balls need to be removed. Or you can use DTAB/CTAB method to process eye stalks with eye balls. For samples from smaller animals such as PLs, you don't have to care their eye balls.

Hepatopancrea also contains PCR inhibitors; besides, it is a digestive organ which contains many DNase.  DTAB/CTAB extraction is also recommended in this case.

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Will the DNA concentration affect PCR results? What's the ideal DNA concentration for PCR?

Too much DNA will inhibit PCR by reducing the amplification efficiency and sometimes cause smear problem. DNA concentration from 200 ng to 1000 ng per reaction is recommended.

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