Who can help me?-Molecular Biology

when I extracted DNA from chicken red blood cell,I met a very strange question.After the phenol and chloroform treation.I collected the upper layers (it is not liquid but like some transparent stiky gel).When I add 2V 100% ethnol to it, At first there are two layers the upper one is white and the the lower half is transparent then there are some white powders appeared.I do not know what the white powder is.I extract DNA from fish red blood cell before using the same protocol and did not met this situation.Now my DNA yield is low and quality is bad.

-linhxm-

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Hi,

did you perform a proteinase K digestion (55 ON) before pheno/chloro extraction? Your product looks like large amount of proteins contamination that you can not remove only by solvants extraction. Maybe it will be better.

-scogne-

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Hi,scogne

I have added PK in the lysis buffer ( final concentration is 150ng/ml)and incubated them overnight at 55℃.I think it is enough.

-linhxm-

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Sorry,I made a mistake in the last thread.The final concentration of PK is 150ug/ml.

-linhxm-

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Hi,

i use to extract genomic DNA from different source by following this procedure:

- lysis and RNase A treatment 2 h @ 37℃ (vol=700? ie)

- add 30 ? PK 10mg/ml and incubate 55? ON.

- phenol/chloro extraction 1h @ 4℃ under soft rotation.

- chloro/isoamyl alcool (24:1)

- 1 Vol Isopropanol precipitation.

Maybe I use a higher conc of proteinase K than you...I don't know, but I  think it is a protein contamination.

-scogne-

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Thank you very much.I have know it is protein now and have treat the DNA samples using PK again and the result were good.

-linhxm-

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The white powder is DNA but it got fragmented. Keep everything the same but include 0.25 M NaCL in the aqueous phase. You will not get the white phase or powder when you add ethanol.

Good Luck

Sam Brilliant

Ph.D. Rescue Team

A division of DSTF-Global

-SamBrilliant-

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