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 estimate the mass of DNA you are purifying according to the brightness of your band on a gel in comparison to the DNA ladder. Actually, using more QIAEXII hurts a lot as it binds DNA strongly and you loose your DNA during purification mostly because it is hard to elute DNA from the beads (it's always better to increase the elution time and perform it that at 50-60C even if the protocol doesn't say so). I use several times less QIAEXII than recommended in order to increase the yield.
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!
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.
You can estimate the mass of DNA you are purifying according to the brightness of your band on a gel in comparison to the DNA ladder. Actually, using more QIAEXII hurts a lot as it binds DNA strongly and you loose your DNA during purification mostly because it is hard to elute DNA from the beads (it's always better to increase the elution time and perform it that at 50-60C even if the protocol doesn't say so). I use several times less QIAEXII than recommended in order to increase the yield.
Yes the μg DNA can be calculated roughly comparing it to your ladder. Match the DNA band with the one in ladder showing same intensity and calculate it according to the loaded volume. Honestly, the kit with beads it little tricky to work with. I prefer the Qiagen one with columns, that's better in terms of yield and output.
Vasundhara Thakur , Anastasia Fokina thank you for the reply. So, i try to match the dna band with the one in ladder. Below is the image of my dna band and the 1 kb ladder. So, if i match it with the ladder that show same intensity, the dna mass will be 92 ng/5 μL, so it means 18.4 ng/1 μL.
As i used only 3 μL ladder, so 18.4 ng x 3 μL = 55.2 ng /3 μL.
Then i have to convert it to μg, so 55.2 ng = 0.0552 μg, which means it is ≤ 2 μg DNA, so i just need to add 10 μl QIAEX II to the sample. Is the calculation correct? Thank you once again for the help.
Dear Nur,
You can compare your DNA band to the ladder only if the size of the DNA fragments and the intensity of the bands are similar. On your image they are not similar and your band is way brighter than any band of the ladder. The calculation itself is correct (if I understood it) but definitely the concentration of your DNA is few times higher that you think. However, the amount you've loaded is not enough for the isolation.
You also have a problem with electrophoresis. I can imagine that your PCR-product can look like this because of the high concentration or due to the reaction itself, but the ladder's bands should be sharp, something is wrong there.
1) Run reaction in ~100 ul;
2) Make a good gel of proper concentration, load a bit of your reaction so you could compare the brightness with the ladder, run not to fast;
3) Calculate the concentration of the product in your reaction;
4) Load the whole reaction on the gel with a big well(s) (100 ul, or 2 x 50 ul);
5) Cut the band out and purify DNA (it is easy to find out how much is there knowing the concentration you've calculated).
Hey Nur, your band does look like ≤ 2 μg. Its 18.4 ng/1 μL, so you multiply it with your PCR sample volume loaded to get your final concentration. But it is just a vague idea to estimate the concentration. If you are eluting this band then keep the elution volume low. I guess they specify 50μL volume, keep it to 15-20μL instead. Depending on your purpose of the elute, if you need more of it then definitely proceed with more PCR reaction
volume to get better elution results.
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