disappearing vector after digestion - (Jun/19/2006 )

I just have incubated this sample and another vetor in the cutting buffer for 1 hour at 37 and they showed up. Then i cut again, purified and nothing...

A260 says there is 19.5ng/ul in the sample, so it should show on the gel.
There cannot be a nucleases in the vector, because i used it straight out of the kit from NEB.
Have tried cutting with one or two enzymes as well without any difference, so that´s not it either.

The restriction enzymes are EcoRI and SalI
[/quote]

I don’t get it, mmm, If when you digest you see the right bands, you said you cut and pudified... the problem could be in the band purification. How do you purify your band???

[quote name='aztecan princess' date='Jun 21 2006, 03:17 AM' post='56333']
I just have incubated this sample and another vetor in the cutting buffer for 1 hour at 37 and they showed up. Then i cut again, purified and nothing...

A260 says there is 19.5ng/ul in the sample, so it should show on the gel.
There cannot be a nucleases in the vector, because i used it straight out of the kit from NEB.
Have tried cutting with one or two enzymes as well without any difference, so that´s not it either.

The restriction enzymes are EcoRI and SalI
[/quote]

I don’t get it, mmm, If when you digest you see the right bands, you said you cut and pudified... the problem could be in the band purification. How do you purify your band???
[/quote]


The problem is solved. On the fourth attempt i could at last see a band! Now I can be happy for ten minutes, before something new goes wrong!