DNA yields with the Qiagen PCR gel purification kit - (Aug/23/2007 )
Hi,
what are the experiences with the DNA yield from the Qiagen PCR gel purification kit? My yield has so far not been very good, about 1/10 of the input DNA
What can I do to improve the yield?
Thanks
I consistently get around a 25% yield from this kit, which is not great, but I can work with it.
I assume you have added isopropanol as suggested in the protocol, if it is appropriate to your size of DNA. Also, if doing the gel extraction step in QG buffer, I would incubate for 30min at 50°C to enure the gel slice is completely dissolved.
Aside from that, I find that the main determinant of the yield is the elution step. I normally use 50ul of EB to elute because concentration is more important than overall yield for my experiments, but I have found that using 100ul of more in the elution gives a better overall yield, although the concentration can obviously be lower. It is also sometimes useful to use hot EB in the elution step.
For all Qiagen kits I elute in two steps: add 1/2 buffer incubate for 1-5 min and centrifuge as it says in the protocol, then repeat.
try invitrogen gel purification gel. we have found it better than Qiagen.
Try to use 65C water to elute DNA. Use 30ul and you would have a concentrated solution.
Incubation with the elution buffer helps.
I generally lose about 90%. I'll try what others have mentioned.
With QUIAquick gel extraction kit???
I think you’re doing something wrong, may be your kit is too old or something like that.
What’s the size of the band???
If it’s too small or too large (more than 8 kb) the efficiency decrease.
To my 40-50 kb band I use QIAEXII with very good results.
thanks everyone.
One thing interesting is that our kit is old (abt 2 years).Maybe its time to buy a new one.
technico-commercial guy told me that binding buffer and elution buffer are quite stable over years. The washing buffer changes little. So you may reprepare some solution of it. I tested once, readding few ethanol gives back good yield. (was on my old nucleospin kit, i considered the solution as 60%EtOH and add amount to get virtual 70%)