restriction digestion problems - digestion problems with SalI (Mar/14/2006 )

Hi everybody,

i have problems with digestion of genomic poplar dna with salI and also a bit pstI, is there everybody who has also these problems or can help me.
I make a southern analysis with 10µg DNA in a total volume of 100µl and 50-70 U Enzyme ,O-buffer 10 fold (Fermentas) overnight at 37°C.

Thanks

Haui

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Your DNA concentration sounds quite high. I would recommend diluting your reaction, either with less DNA or with higher volume. I would aim at 20 ng/ul. SalI is known to be fussy. You might also be seeing methylation: SalI is blocked by CG methylation of the central CG in the recognition site.

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Hi phage 434,

i think it is usual to take 10µg DNA for a southern analysis of plant material. I agree with salI.
I will not take salI and also not pstI, therefore i take speI,ECoRV, HindIII and XbaI. These are the only enzymes i can take because of my cutting sites in the construct.

what you are mean to these enzymes.

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