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restriction digestion of genomic poplar dna - digestion problems with genomic dna of poplar (Mar/10/2006 )

Hi everybody,

i am trying to make a digestion of poplar genomic dna with the enzymes salI,pstI,speI,hindIII,xbaI and ecoRV
after the digestion overnight 37°C the dna is not good digested, salI function not good, the control with undigested dna is also a smear on the gel, after dna isolation the dna looks fine on the gel
The reaction was made in 200µl h20 sterile,10µg DNA, 80U Enzyme and buffers (fermentas, with bsa in it).

maybe the water is bad? the dna is not clean, sometimes after the dilution in H20 there were some pieces in which are not dissolving, spec was ok 1,8-1.9 260/280 ratio
i isolate the dna with nucleon phyto pure from amersham

What do you mean, i am trying to make a southern analysis of my transgenic poplar lines.

Thanks

haui sad.gif

-haui-

I'm not entirely clear what your doing, but...

If you're digesting with these enzymes together you may not get a sufficient digest. I didn't look up the specifications of your enzymes by they will work differently in the same reaction, especially if they require diffrent buffer conditions. Your salI result may be an indicator of buffer incompatibility.

Also, if you do the math for each RE recognition site to estimate the size and number of fragments, you're going to get a smear... You'll probably see a smear with only one enzyme as well because of the variablity of the sequence.

You can try a sequential digest, but with that many enzymes it would be very difficult.

-vasussci-

QUOTE (vasussci @ Mar 10 2006, 09:34 AM)
I'm not entirely clear what your doing, but...

If you're digesting with these enzymes together you may not get a sufficient digest. I didn't look up the specifications of your enzymes by they will work differently in the same reaction, especially if they require diffrent buffer conditions. Your salI result may be an indicator of buffer incompatibility.

Also, if you do the math for each RE recognition site to estimate the size and number of fragments, you're going to get a smear... You'll probably see a smear with only one enzyme as well because of the variablity of the sequence.

You can try a sequential digest, but with that many enzymes it would be very difficult.



I want to make southerns with my transgenic poplar lines to determine the number of transgene copies in it. More not.
I do not digest with all enzymes together. I have digestions reactions with 4 enzymes separately and one control undigested. every reaction has only one enzym with special buffer and 10µg dna.

what i see now, is that the dna after the isolation is not fully dissolved in TE, the pellet is brown after washing. Maybe the kit is not good. i will try it in a classical way with CTAB and PHE/CHL/IAA ....

haui

-poplar-

Did you try to add the enzyme, put to incubate and after 20 minutes add another bit of enzyme?

-Dees-