Low copy number of plasmid - (Nov/27/2004 )

Dear all

I have ligated DNA target (that might be toxic for cell) to pGST-4T-2 plasmid. But, copy number of the recombinant plasmid was very low. Does anyone can help me how to get the enough amount of the plasmid after mini preparation.

Thank

Hi
1. u can try to grow cells in terrific broth
2. check the insert size limitations of the plamid
3. ensure your selction pressure conc. (antibiotic)

ravi

Dear Ravi

Thank a lot
I cultured plasmid-bearing E. coli in 4 ml of LB medium (with 4 mikroliter of 50 mg Amp). The size of insert is 200 bp.

Try 10 ml culture! It works most of the time fine... Then elute with warm buffer/water (I use 30 °C) - this will increase the yield.