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how to increase DNA amount about 10 ug for digestion

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how to increase DNA amount about 10 ug for digestion

Postby biolog35 » Fri Jun 20, 2008 7:29 am

I have a bacterial strain which is pseudomonas.I tried to construct a genomic DNA library.for that ı have to do partial digestion and ı need at least 10 ug genomıc DNA but ı couldnt get this amount.I am using a manual procedure (protocol in current protocols in molecular biology) .How can I increase my DNA amount? (DNA amount:90 ng /ul)
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Re: how to increase DNA amount about 10 ug for digestion

Postby Fruitfly » Fri Jun 20, 2008 8:50 am

I think you need 1000/90 microlitre or you have to isolate more to gain amount you need
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Re: how to increase DNA amount about 10 ug for digestion

Postby biolog35 » Fri Jun 20, 2008 12:46 pm

ok but ıt ıs too diffucult u know.ı thınk there must be another method.thaks:D
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Postby canalon » Fri Jun 20, 2008 2:46 pm

So you have less than 12µl of your final DNA prep? That sucks.

Solutions:
1-Use a different and more efficient method (CsCl is not fun but allow purification of tons of good quality DNA). And you might try to improve cell lysis, I do not think that Pseudomonas are easy to break open.
2-repeat until you have enough and concentrate in one tube (EtOH precipitation, glycogen addition possible, or simply use a speed-vac)
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Re: how to increase DNA amount about 10 ug for digestion

Postby biolog35 » Sat Jun 21, 2008 12:22 pm

thanks canolon:D but CsCl METHOD İS efficient method ı know but it takes more time and requires large amount of reagents.it is a second solution for me now .yes u are rıght pseudomonas is diffucult to lyse and also produce large amounts of polysaccharides so CTAB method is suitable for me.also ı'll try to concentrate my DNA solution after serial isolation.also there is another problem .by usıng CTAB method my DNA solutıon also ıncludes RNA .I REMOVE İT wıth RNAase treatment and ı have to remove thıs enzyme from my solutıon by phenol/chloroform extractıon that also causes lost of DNA :? IS THERE ANOTHER OPINION?
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Postby canalon » Sun Jun 22, 2008 2:08 am

YEp no technique is perfect. You can improve recovery of DNA during precipitation by adding glycogen as a carrier (Id onot have a link handy, but a research should not be difficult to find what amount of glycogen to add). Using isopropanol, or butanol is also supposed to improve DNA precipitation efficency.

I do not know any method to avoid DNA during the CTAB and phenol chloroform steps. Just to be very careful.

Another solution might be to use DNA maxi or midi kits. But I have not always be happy with the consistency of the results.
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Re: how to increase DNA amount about 10 ug for digestion

Postby biolog35 » Sun Jun 22, 2008 1:51 pm

thanks canalon :D ı ll try tomorrow all things we discuss see u
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Re: how to increase DNA amount about 10 ug for digestion

Postby Fruitfly » Fri Jun 27, 2008 12:59 pm

to biolog35:why don't you treat your cell extract with RNase before adding CTAB to cell extract ?
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Re: how to increase DNA amount about 10 ug for digestion

Postby biolog35 » Sat Jun 28, 2008 2:44 pm

ı trıed ıt fruitfly but it did not work:(
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Postby canalon » Sun Jun 29, 2008 1:50 am

If I remember correctly the lysis buffer contain Guanidine isothiocyanate (GTIC) or another chaotropic agent that denaturate all the proteins, thusprotecting DNA and RNA. Basically you cannot treat with a protein before the precipitation and cleaning of the nucleic acids, because all proteins would be denatured by the buffer.
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Re: how to increase DNA amount about 10 ug for digestion

Postby biolog35 » Sun Jun 29, 2008 10:00 am

but my protocol does not ınclude thıs buffer:s ı lyse my cells by ıncubatıng them sds and proteınase K.
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Postby canalon » Mon Jun 30, 2008 3:39 pm

Ok. Well I guess the proteinase K would not be very friendly to your RNAse either, would it? Besides, even of the RNAse is not degraded I wonder if SDS and other conditions are optimal for RNAse activity?

As far as I can see, either you can find other degradation buffer to improve your conditions (you can for example adapt the lysis conditions used in this paper for PFGE, with probably shorter incubation time, since you will not need to pellet your cells in agarose: Indian J Med Res. 2007 Aug;126(2):146-51, free full text available through pubmed). Or you scale up, make many repeats of your extraction until you have enough DNA.
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