Help: I am loosing the DNA during PCR purification - (Sep/27/2011 )
Dear fellows, it was not used to happen but Im currently losing the DNA when I try to purify my PCR products through columns. I have already tried two kits (Illustra GFX PCR DNA and gel band purification kit - GE healthcare; and Megaquick-Spin PCR & agarose gel DNA extraction system - iNtRON Biotech) and the result are the same. Recovery from neither direct PCR products nor excised agarose gel bands were well succeeded. It is noteworthy that the PCR product appears in a ~1,3Kb sharp and bright band. Could anyone give me some clue?
I'm not specifically familiar with the iNtRON kit, but I have used multiple Illustra kits and have found a lot of variability, even within the same box I have had columns that worked really well and columns that did not work at all. Many of these extraction kits have pH dependent binding processes and it is possible that your buffers have gone off. I would call the company and ask them to send you a test kit (they usually have samples they give out free) from a different Lot# from the one you currently have. Test the fresh kit against your old one to see if your buffers or columns have gone bad.
Alternatively, I routinely use the QIAquick purification columns and I have never had the problems I had with the Illustra kit.
Best of Luck.
Are the kits new? if they are quite old, may be the ethanol in the wash buffers had been evaporated and you are eluting the DNA in those steps...from my experience acquiring a new kit is always a good idea and cleaning PCR products give always better yields than the gel band extraction...
What is the proximate DNA concentration that one commonly get after PCR purification?
between 1-3 ug in my case...
I don't really go off of concentration since it is very dependent on input concentration. If my PCR worked really well and I have 5 ug of product (in a 50 uL rxn), I will usually get back around 3.5 - 4 ug (using QIAquick PCR cleanup) which is about 70%-80% yield. For less concentrated PCR reactions (around 0.5 - 1 ug) the yield is usually lower, around 50%. Your final concentration will alway depend on how much buffer you elute in, and some kits have different ranges so if you are looking for a certain concentration range, pay attention to what your allowable elution volumes are.
Best of Luck.
i am using MegaQuick iNtroN kit ... & it works verry fiinee with me ..
1) i check for the buffer pH ( by observing no change in color ).
2) i add isoprpanol.
3) i do twice washing steps.
4) i add finally only : 15ul elution buffer.
all the good luck.
I use MegaQuick iNtroN kit as well.. but you have to be careful about the column... sometimes it will not function well (leak) esp you are loading a higher concentrated melted agarose....