double digest problem - (Jan/28/2012 )
i did a double digest with Ecor1 & BamH1 with a 7Kb plasmid sample prepared through miniprep but not purified.
in 10ul reaction volume i used 2 ul DNA ,1ul10xBSA ,1ulNEbuffer4 and 0.2ul of each enz.
got no expected fragment . these were plasmids taken from transformants of the ligation mix of backbone + fragment.
Many reasons :
May be the ligation didnot work. Did you mix the enz. tube before pippeting 0.2ul out. The amount of fragment that you wish to see could be too low as the starting material is also too low, 2ul of miniprep DNA.
Better to do a digest in a larger volume, say atleast 20ul.
thanks a lot. i did not mix the enzym. yes even i think when i had done 20ul rxn for gel purification i had got a fragment but that was for the original mutant plasmid whose fragment i wish to sub-clone. in 20 ul how much DNA do i take
You need to take as much DNA so that after the digest the fragment, which is a proportion of the total DNA, is visible on a gel. If the fragment is too small, then you need to have more DNA than if it was big.