Dear all,
As a newcomer, this is my first time to ask for help about my research projects. please say all you know about my question. Thanks very much.
Now I am busy with the isolation genomic DNA from the chocolate, but the results is very poor, the DNA elution is lightly yellow and no target band can been seen by the agarose gel. How to deal with the though question? Do the every member of the researchgate have a ideal method to resolve the problem?
best wishes
Yunjing Zhang
Hey Yunjing!
Are you using a Kit or are you following a certain protocol? It's difficuld to make suggestions, when we don't know anything about the procedure..
Cheers!
Hi, did you check the concentration of isolated genomic dna? maybe its too low. One reason could be you might be adding high volume of dh20 at elution step. In final step elute you DNA in very less volume of elution buffer or dh20 (e.g. 15-20ul). It would be better if you pool DNA from several isolations (5-6 tubes). If your nest step is PCR you can try PCR even if DNA is not visible on gel as PCR will amplify the gene of interest even if template is in low concentration (depends on which brand PCR mix you are using read its manual). Hope it helps.
hallo, check Gopal Tiwari proposal. But it looks like, that you have problems with your isolation method itself. Receiving colored solution shows you, that you isolated not only DNA. Coloring substances often contain double charged ions that disturb or inhibit the PCR reaction. Therefore try other isolation methods.
In addition: do you use real-time PCR or conventional PCR (you mentioned Agarose gel)?
A good kit. Another well performed and similar in use is the "Macherey-Nagel food kit", used in my lab for a wide range of samples, including highly processed food or food suplements like gelatine and liquid lecithin. But it makes sense to compare two or three kits regarding their DNA-yield and -purity. Sometimes there is no way to use the extensive CTAB method, that often produces only small yields of DNA.
You could try this protocol, which is very useful for plant total DNA extraction.
3% CTAB DNA extracion protocol
(modified from Zeng et al. 2002, ACta Botanica Sinica 44; 694-697)
1. washing out polysaccharides with CTAB-free buffer from homogenate.
2. increasing the percentage of CTAB in extraction medium.
3. using high concentration salts pior to DNA precipitation with isopanol.
Please see following publication on comparative Evaluation of Six Extraction Methods for DNA Quantification and PCR Detection in Cocoa and Cocoa-Derived Products
http://www.tandfonline.com/doi/abs/10.1080/08905436.2014.996761?journalCode=lfbt20
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