Plasmid Purification - Any tricks to increase yield? What could I be doing wrong? (Jun/04/2006 )
I am trying to purify a plasmid and digest it so that I can clone the gene into another vector. We have two kits in our lab:
Mo Bio Laboratories UltraClean Mini Plasmid Prep Kit and the Sigma GenElute MIni Plasmid prep kit.
I have primarily been using the Mo BIo kit but seem to get a very low yield, even when my supervisor has purified from the same culture and gets a much higher yield than me.
I have seen advice (I think it was for a gel DNA purification kit) saying that if you heat up the elution buffer to 65C you would get an increased yield. Is this also true of the plasmid purification kits?
Any other advice that may increase yield, or any suggestions of what I could be doing wrong?
thanks in advance for your assistance
i do an alkaline lysis protocol which goes very good in my hands. PM me if you want it.
For kits, i allow the column to air dry (or 37° dry) for at least 30'. Better recovery for sure.
I also heat the elution buffer when eluting plasmids to 70°, and for plasmid purif i elute in 2 times.
If i don't have enough plasmid (i keep the column till OD reading), i redo an elution and concentrate with butanol.
i found some useful tips here.
i think a couple of things to try with columns to increase yeild - 1) make sure that you pipette your elution buffer directly onto the matrix rather on the side or something and 2) i generally leave the elution buffer on the column for 5 min or so before i spin down.
Also, are you and your supervisor both eluting in the same solution?.....