Problems after site directed mutagenesis - (Jan/11/2008 )
Hi all,
excuse my english, i'm a german student. My problem:
i've trying to do a site direct mutagenesis, then transform the pcr product after digesting with dpn1. Afterwards i made mini plasmid preparations (kit - macherey-nagel). Analyzing the samples with a agarose gel shows the plasmid in the right band. But whether I make a restriction with one enzyme (linearisation) or with two enzymes to cut out the insert i doesn't see anything!!
Perhaps someone knows the problem or has any idea what has happened.
Hi
Are you sure that you have the rigth plasmid??? After your PCR transformation did you use an antibiotic resistance???
It could a contaminant plasmid ... or it could be your restriction enzymes...
excuse my english, i'm a german student. My problem:
i've trying to do a site direct mutagenesis, then transform the pcr product after digesting with dpn1. Afterwards i made mini plasmid preparations (kit - macherey-nagel). Analyzing the samples with a agarose gel shows the plasmid in the right band. But whether I make a restriction with one enzyme (linearisation) or with two enzymes to cut out the insert i doesn't see anything!!
Perhaps someone knows the problem or has any idea what has happened.
what amount of digested DNA did you load on gel. Try to have sufficient DNA to visualize it on gel. Also run the undigested DNA side by side as control.
excuse my english, i'm a german student. My problem:
i've trying to do a site direct mutagenesis, then transform the pcr product after digesting with dpn1. Afterwards i made mini plasmid preparations (kit - macherey-nagel). Analyzing the samples with a agarose gel shows the plasmid in the right band. But whether I make a restriction with one enzyme (linearisation) or with two enzymes to cut out the insert i doesn't see anything!!
Perhaps someone knows the problem or has any idea what has happened.
could you be more specific - are the restriction enzyme sites ones that you introduced by site directed mutagenesis,? or are you just trying to cut with sites that were already there?
Hi,
thanks a lot for your answers.
@ Artero: Yes I used an antibiotic resistance (Amp) after my PCR transformation.
But I thought, if the restriction enzyme is out of order the result would be a non-cut plasmid. Star activity comes from the wrong buffer and to much restriction enzyme in the digestion, or?
@ Scolix: After the first empty gel (I could only see the Marker) I analyzed always the undigested DNA together with the digested DNA on the same gel. The amount of the digested DNA and the undigested DNA were always in the same range: 1,0-1,5 μg
@ Smu2: I didn't try to introduce new restriction enzyme sites, I tried to cut with the sites that were already there.
Do you see a smear or anything thing to indicate DNA is degrading? Could you try to precipitate the DNA and resuspend it in new TE buffer and then digest it.