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contamination

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contamination

Postby sara » Thu Apr 28, 2005 9:36 am

hello
im a new member. my quastion is how can i know if my extracted DNA from plant contamination with RNA or polyphenols ? and if it contains one of them this will effect if i do PCR and RE?.
sara
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Postby DevGrp » Thu Apr 28, 2005 11:20 am

If you are worried about RNA contamination include RNAse in your extraction procedure or incubate your DNA with RNase.
Polyphenols and polysaccarides are harder. Depends on the plant and the extraction procedure.
Try a control PCR reaction / digest and spike it with some of your extracted DNA if this inhibits the PCR reaction / digest you have a problem

What plants are you working with and how did you extract them?

We have been testing out various extraction procedures for getting DNA out of Hemp and Artemisia. At least one method gave good looking Artemisia DNA but it was impossible to PCR from it.
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Postby sara » Thu Apr 28, 2005 9:36 pm

hi DevGrp,
im working with wild plants using many extraction methods some like Doyle & Doyle , Dellaporta et al, Rogers and Bendich methods which they are using chloroform:isoamylacohol.

not all samples containg RNA or polysacchrides but some like Boraginaceae and compositae family.

im used RNase before but the concentration of DNA become vary less which then i can not use the same sample for RE.

There is other things can i use it than RNase for RNA treatment ? and if im using RNase how can i clean sample from it?.
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Postby sara » Fri Apr 29, 2005 7:42 am

yestrday im do the PCR and RE to the sample which contain RNA and polysacchrides and this sample was amplified and digested, so the contamination are not effect my work.

i want to know when the RNA and polysacchride can block amplification and restriction? may be if it in large quantities?
sara
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Postby DevGrp » Fri Apr 29, 2005 12:18 pm

The simple answer is if your experiment and suitable controls work then you don't have a problem. I'm not really sure how much contamination is a problem, if it works don't worry. If it doesn't work , try a different extraction method. For most applications the RNAse still being present isn't a problem. It depends on what you want to do with your DNA next?
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Postby sara » Fri Apr 29, 2005 1:48 pm

thanks to all reply DevGrp


regards
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