You must have a isolation protocol, as far i know if you have good primers ploidity should not be a problem. You are not the first person working with. It tells you just the copy number of identical chromosomes. Isolated DNA should be clean.
Well , check your primer again but it is always better to have two different RE . You do not have a direction problem or multimerization of insert. Furthermore, your insert and vector is really big in size, so check again different ligation condition. The big colonies mean that your CIAP reaction was not completly successful.
Q5: It seems that NdeI is having some difficulties cutting my DNA. Is there a reason for that?A5: NdeI activity is sensitive to contaminants present in DNA isolated with standard miniprep protocols. Further purification of the DNA by a column or dialyzing the contaminant from the DNA may help increase the activity of the enzyme on the isolated DNA substrate.Q6: NdeI seems to be digesting my DNA correctly, but when I try to ligate it, I obtain no colonies. Is there a potential explanation for what I am observing?A6: NdeI is a very robust enzyme. If the substrate DNA is digested for extended periods of time, we have found evidence that the enzyme will remove some additional nucleotides. Digestion of DNA with NdeI over 4 hrs is not recommended.
Maybe your gene is multiple cloned into the vector.And using 2 or 3 colonies is pretty few. And your primer might be not specific enought.
Lets say İ would like to clone my Gene BamHI and Not1 into pGEX GST N. When İ do that i will get additional Amino acids on my rProtein after cleavge with thrombin. İ think gly will not effect my overall structure but nevertheless its there.
İ have a stupid question. İf i clone a HİS tag directly to my rProtein İ have no Amino Acid inbetween. But in case of GST Tag İ am getting additionally AA to my rProtein. Am İ right? How can İ avoid this.