I have carried out a PCR reaction, resulting in a product of ~227bp. I quantified the raw product using PicoGreen, while this isn't entirely accurate with other components potentially interfering, it resulted in concentrations of ~ 200ng/ul. Gels also showed good bands.
However, after PCR purification with the PureLink kit (using Buffer 2, not Buffer 3) I have been left with 7 ng/ul (Nanodrop figure) and a negative PicoGreen value. I used 23uL of PCR product for purification.
Can anyone help with this drastic loss of product? I tried to re-run the elute through the column a second time, with no difference being seen!
are you sure that the binding buffer had isoprpanol added and that the wash buffer had ethanol added either of which would result in low binding of dna to the column. you could try eluting the purified dna with hot (70c) elution buffer left on the column for twice as long as recommended which does increase yield but the likely explanation is poor binding in the first place
Yes I diluted them myself just two days before use - thank you for the suggestion I will try that for sure!
Hello Charlotte,
from my experience, there is always a tricky threshold with the yield of PCR product purification that I honestly can't explain. Like, the yield you get is linear with your starting material until a point were just dividing the PCR reaction volume you purify by 2 decrease the yield by 10.
I had the same problem with Macherey-Nagel gel extraction kit where my bands were clearly there and like you after elution, between 4 to 8 ng/µL....
After trying multiple time to tweak the protocol with no reproducible improvment I just decided to use brut force.
Just purify starting with a 100 or even 200 µL PCR reaction volume and I garentee it'll work every time.
It's a shame not to know what happens but sometime you need the work done haha.
What are your downstream application for the purified PCR product if I may ask ?
Good luck,
Philippe.
I agree with using a larger amount of PCR volume for the purification. As a note you'll want to set up a set of small volume reactions (20-30 ul) rather than trying to run a large volume (100 ul and up) as the efficiency can be decreased for larger reaction volumes.
Your PCR product is on the small size, double check the size range of the kit you are using in case it is close to the lower limit.
Hi all, thanks a million for the suggestions! I am hoping to carry out basic Sanger sequencing on the products. The company who makes the kit suggested that I can add nuclease free to make up my PCR product to 50uL as the kit works best with higher amounts? I think I'll try it out so fingers crossed!
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