Low yield PCR product after gel purification - (Mar/13/2013 )
I need everyone help and suggestion. I have try to purified PCR product from gel purification kit. I've check PCR product by electrophoresis I found that I got thick band of my target product but after I purified I got very low yield ( 9 ng/ul). So I try another way, by precipitate PCR product first and measure conc. got 1.4 ug/ul. Then, gel purification with kits. Unfortunately, I still got low yeild (9-10 ng/ul). I have more reviewed. I found another way. that's using enzyme agarase for digest gel directly not need use column purification that maybe couse low yield. Everyone, How do you think about this way and have another techniques for gel purification with high yield , I just need 100ng for go next step in my study. Thanks all for reading and suggestion. I'm look forward you helping.
Wow, that seems like alot of work for PCR clean up. Can you modify your initial PCR reaction to produce one distinct band of your target size. From there you could just do an phenol precipitation and not lose any DNA in a column? If not, what is the maximum concentration of DNA that your column can elute? Have your tried modifying your gel extraction protocol? I usually let my elution buffer sit in the column for 10+ minutes, spin and then send my elution buffer back through. This significantly raises my final DNA concentration.
Are you positive your dilution concentrations are correct? What is the final volume of your DNA elute? You do not have enough for 100ng?
I used Purelink kits from invitrogen. Its mention that purify up to 15ug per column. Today, I eluted twice like you recommended but still got 3-4 ng/ul from 50 ul PCR product (2 reactions,25 ul each). Have any idea?
concentration doesn't give yield, total mass does. what is the volume at that concentration?
Dear mdfenko the volume is 4ng/ul in 30ul. Anyway, I want high conc.in 1ul for next round PCR at least 20-30ng/ul.
Gel extractions typically give low yield, 5-20 ng/ul is typical. As Jerryshelly says, you will probably be better off making sure that you have a single band and then purifying that directly from the reaction, rather than trying a gel extraction.
Also note 4 x30 = 120 ng... You could try concentrating the DNA by evaporation.
Another option is to do several PCRs and then pool the products together.
Are you doing gel extraction of you PCR product? Or just seding your PCR reaction mix through the column?
Column clogging is one of the major reason for low yield. So cut the DNA band avoiding gel as much as possible. Make sure you dissolve well all the remaining gel from the given buffer. If any of the buffers have ethanol/isopropanol, let the column dry for few minutes before proceeding. Any residual ethanol will retain DNA on the column rather than eluting. Let the elution buffer to stay in the column for ~5 min before centrifugation.
I had the same problem with Quigen kit and after doing so many trials I managed to increase my product concentration upto ~100ng/uL.
Good luck !!!