messed up restriction sites - (Apr/10/2009 )

I have a problem! I cut my vector (pBluescript SK+/-) with SalI/ClaI, add my insert, and ligate. I get insertion, but every time the ClaI site is no longer usable. Does anyone know why the ClaI site keeps mutating (I checked the sequence and it is changing from ATGGAT to ATGGAA or ATCGTC, etc? I need to use this again for another insert!

lnorwood on Apr 10 2009, 01:41 PM said:

Are you sure that the sequence is mutating every time? Have you checked your insert primer sequence?
Also, the recognition site for ClaI is ATCGAT, which is not a substrate for Dam methylase; however, if that sequence is followed by a C or preceeded by a G, a Dam recognition site is generated and cleavage by ClaI is inhibited (http://www.vivo.colostate.edu/hbooks/genetics/biotech/enzymes/cuteffects.html).

So, I am cutting my insert from a TOPO TA vector. I know my insert was made correctly because I sequence verified it.
As for the cutting of the insert and vector, I am growing my plasmids in SCS110 (dam/dcm deficient bacteria), so whether there is any nearby methylated site is a moot point. These plasmids are not methylated anyway.
I used specific primers to my insert to sequence to verify my cloning, and that is when I find that the ClaI site is messed up (how it's changing, I do not know).
And I mis-wrote the recognition seq for ClaI before. Yes, it is ATCGAT. The two clones I have, the sequence is changed to ATCGAA or ATCGTC. Does anyone understand why this would happen?



Are you sure that the sequence is mutating every time? Have you checked your insert primer sequence?
Also, the recognition site for ClaI is ATCGAT, which is not a substrate for Dam methylase; however, if that sequence is followed by a C or preceeded by a G, a Dam recognition site is generated and cleavage by ClaI is inhibited (http://www.vivo.colostate.edu/hbooks/genetics/biotech/enzymes/cuteffects.html).