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Digested DNA cleaning - My DNA is in dh2o and I need it to be in TE for the phenol/chloroform (May/28/2007 )

Hi,

This may be trivial, but I have a plasmid DNA sample that I will be linearizing. It is in dh20 and I would like to know what concentration of TE buffer (1X,10X,100X) is best before initiating a phenol/chloroform extraction to clean off any digestion reminants. I have TE saturated phenol that I plan to use for the extraction if this info helps.

Thanks a lot!!

-Embryos-

1x would be the standard thing to do, but you could work directly with the water solution. The main difference between 1x TE and water is the pH (7.5) and the presence of EDTA, which chelates any magnesium present. The pH is important in dissolving DNA, which is much easier in slightly alkaline buffers. The EDTA is important for long term storage, especially at RT or above. Since you are purifying the DNA and it is already in solution, neither of these apply. When you are done and ethanol precipitate your DNA, I'd recommend resuspending in 1x TE for storage.

-phage434-

phage is correct. Just use 1 x TE buffer will do.

-timjim-

I agree with others about using 1x TE.

I would run a gel of the digested DNA and purify it thru a column and elute it with TE.

-scolix-

QUOTE (phage434 @ May 28 2007, 05:59 PM)
1x would be the standard thing to do, but you could work directly with the water solution. The main difference between 1x TE and water is the pH (7.5) and the presence of EDTA, which chelates any magnesium present. The pH is important in dissolving DNA, which is much easier in slightly alkaline buffers. The EDTA is important for long term storage, especially at RT or above. Since you are purifying the DNA and it is already in solution, neither of these apply. When you are done and ethanol precipitate your DNA, I'd recommend resuspending in 1x TE for storage.




Thanks! Will put your recommendation to use.

-Embryos-

i use water to dissolve the mini prep DNA that i make to check my desired plasmid

-T. reesei-

QUOTE (Embryos @ May 29 2007, 06:28 PM)
QUOTE (phage434 @ May 28 2007, 05:59 PM)
1x would be the standard thing to do, but you could work directly with the water solution. The main difference between 1x TE and water is the pH (7.5) and the presence of EDTA, which chelates any magnesium present. The pH is important in dissolving DNA, which is much easier in slightly alkaline buffers. The EDTA is important for long term storage, especially at RT or above. Since you are purifying the DNA and it is already in solution, neither of these apply. When you are done and ethanol precipitate your DNA, I'd recommend resuspending in 1x TE for storage.




Thanks! Will put your recommendation to use.



Molbio grade water has helped a lot if you use DNA for downstream processing like restriction digestion

-buddie-