pcr purification - (Aug/16/2012 )
, i need ur suggestion,, i am doing my master thesis , I had my perfect pcr but at the time of pcr purification my supervisor is asking me to use only the volume that can hold only 10µg, as it is mentioned in the pcr purfication kit of qiagen, but i really do not understand why cant I purify the whole product...can u give me a sloid reason so that i can convience my supervisor to do what i want..
And the last time also they asked me to do the purification in the same manner and did not work...and again they are asking me to do the same, but I dont want to do it.. kindly reply me soon..
thanks and regards
Take the entire PCR product and put it on only one column. You will never get out of a PCR the limit of the silica column. After you do the PCR, you cannot measure the concentration of DNA, without cleanup because you have also dNTPs there (which also absorb like the DNA does). So this is why your supervisor might think you have more than 10ug. You will never-ever get 10 ug out of a PCR reaction... unless you pull together several PCR tubes. I usually get out a PCR (20 uL) + cleanup 0.4-1.5 ug.
He is right to be afraid to overload the column. That column has indeed a limit. However, if you overload the column, that means you lose some of the PCR product, but 10 ug still remains there. What happened to you, most probably was over-dilution of your PCR product below the detaction limit.