all the DNA gone after digestion - (Feb/28/2008 )

after double digesting with Bamh and NdeI all my dna was gone
1. BSA(10) 3ul, buffer 3 3ul, dH2O 12 ul, DNA 5ul, Bamh 1ul, NdeI 1ul
2. BSA(10) 3ul, buffer 2 3ul, dH2O 12 ul, DNA 5ul, Bamh 1ul, NdeI 1ul
3. BSA(10) 3ul, buffer 3 3ul, dH2O 12 ul, DNA 5ul
4. BSA(10) 1ul, buffer 6 1ul, dH2O 6.5 ul, DNA 2ul, Bamh 0.5ul, NdeI 0.5ul

37C, 1h

after running the gel, I can only find band in 3, which is the control

it's really hard for me to figure it out, because I already changed the amount in 4, and changed different buffers,
and I reduced the digestion time to 1h, so what happened to my DNA? I really need help here. Many thanks!!

plus, I added the BSA buffer and ddwater first, DNA second, and added the enzyme at last

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Try to do two separate digestions (one with BamHI and one with NdeI) rather than doing a double digestion. Keep all other parameters the same, but use the optimum buffer for each enzyme (Note that BamHI has star activity) to see if one of the enzymes is causing you the problem or if it's the double digestion that does.

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i will try to digest seperately.....

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i tried single digest this time, and i got bands
but i cannot tell whether my dna is cut by the enzyme because it is supposed to be cut 10bps.
anyway, my dna survived at this digestion
i will digest with the other RE and to see the results
thanks for advice

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i have another question here
why the double digest doesnt work, but single digest work?
is there any reason?
THANKS

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again
after the second digestion
all the dna disappeared
someone help me
many thanks

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perhaps your second enzyme became contaminated with DNase at some point. Try the same enzyme from another lab or order new enzyme.

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another guy in our lab used that enzyme too, and it works
so i think its not the problem of this enzyme

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i tried again with the sequence digest
this time i use the Ndel to digest first , and finally i got a faint band, anyway its a band
then i gel extracted this band and stored it in -20C for a nite(cause its too late)
this morning i digest this dna with another RE(Bamhl)
AGAIN my dna disappeared

as i have mentioned , the Bamhl should be fine
then what happened here? is it inappropriate to store the half digested dna in fridge for a nite?
confused
can anyone help me?
many thanks

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