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Problem with Bgl II Sal digestion

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Problem with Bgl II Sal digestion

Postby genetherapy » Fri Nov 18, 2011 11:10 pm

Hi,
I need help about my experiment. I want to digest my plasmid with Bgl II and Sal I. I used the following ratios:
15.8 mikroliter plasmid DNA,
2 mikroliter Buffer3,
0.2 mikroliter BSA,
1 mikroliter Bg I II,
1 mikroliter Sal I
I could not get the digestion bands
Although I digest the same plasmid with Xho I & Bgl II and Xho I & Sal I with same ratios it worked. So the problem is not the enzymes and the plasmid. I could not understand where the problem is?? Please give some advise
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Postby JackBean » Mon Nov 21, 2011 7:56 am

you see no band on the gel or you see the uncut-vector pattern?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Postby cw11 » Tue Nov 29, 2011 2:07 am

Yes, what exactly do you see when you digest with Bgl II and SalI? More fragments than expected, uncut plasmid, plasmid that looks like it was cut only once, nothing...? If one of the first 3, then your restriction sites may be in different places than you think they are...
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Re: Problem with Bgl II Sal digestion

Postby genetherapy » Wed Nov 30, 2011 4:06 pm

I increased enzymes quantitty as belows and it worked. I could not seen any digestion before,only seen the uncut plasmid. I think the DNA was too much for digestion?
13.8 mikroliter plasmid DNA,
2 mikroliter Buffer3,
0.2 mikroliter BSA,
2 mikroliter Bg I II,
2 mikroliter Sal I
I wanted to thank you for your interest and share the solution. May be it will be useful for the other researchers if have a problem like that
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Postby JackBean » Wed Nov 30, 2011 4:32 pm

it there was too much DNA, at least something should be cut anyway. Rather your enzymes were denatured maybe?
http://www.biolib.cz/en/main/

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Postby genetherapy » Wed Nov 30, 2011 4:44 pm

My enzymes are new purchased. The DNA was too much as you told I think. Thank you
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