I'm trying to purify my pcr product by using QIAEX II Gel extraction kit. According to the protocol, firstly i need to add buffer QX1 to the tube (contain gel slice that already been cut) according to DNA fragment size.
Then, i need to resuspend QIAEX II by vortexing for 30 s, then add the QIAEX II to the sample (10 μl QIAX II for ≤ 2 μg DNA; 30 μl for 2-10 μg DNA; and an additional 30 μg DNA).
I didn't understand what is the measurement (μg DNA) about? does it refer to the weight of DNA that have been load during pcr process? If it true, how i'm supposed to calculate the weight? I really hope someone can help me.
you can just use the 'freeze and squeeze' method for gel purification, all it takes is a single filter tip pipette and a centrifuge!
https://www.youtube.com/watch?v=b3wfhofZJSI
Hi Nur,
I think this refers to the amount of DNA present in the gel slice, because the silica particles (QIAEXII) have a limited binding capacity of 5 µg DNA per 10 µl QIAEXII. I know it's hard to estimate how much DNA is in the gel unless you know how much you loaded (and you wouldn't know that when you're purifying PCR products), but I would go with the middle ground and use 30 µl QIAEXII (using more wouldn't hurt). Also, if you can change kits, I strongly recommend using NEB's Monarch Gel Extraction kit. It's much more user-friendly, and you can elute in 6 µl, thus keeping your purified DNA concentrated. Hope this helps, and good luck!
Daad Abighanem I have used the Monarch kit, and it is excellent. But I find with it that if I elute less then 20ul, I lose a lot of the dna. I use a speedvac to concentrate from 30 ul, rather than risk losing dna with low elution volume
Hi Amy Johnson , I agree. I usually elute with 10 µl, and find that, even if I lose some DNA, I still have enough for downstream applications.
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