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  • FAQ: I have done all the standard troubleshooting for my cloning and transfection steps and I still cannot get the desired clone in M13KE?

    Make sure the pIII leader sequence is intact and in-frame with pIII coat protein in your design (see also cDNA FAQ#1). Assuming, the reading frame is correct, the insert may not allow the production of viable phage either due to issues with export into the cell periplasm or function of pIII coat protein after phage assembly. Inserts larger than 25-50 amino acids and those with N-termnal cysteines or positively charged residues are known to be problematic. These issues may be probed by subcloning the insert into another vector with KpnI/EagI sites. For example, try to clone the insert into pMAL pIII (#N8101S) to see if the insert-MBP fusion can be produced and exported to the periplasm.