Dear all,
I amplified a sequence from a plasmid template. The amplicons will serve as templates for IVT. The amplicon is 500bp in size, while the plasmid is about 2000bp in size. I do not wish to have a mixture of my plasmids and the amplicon for IVT. Hence, I electrophoresed the products. Sharp, clean bands representing DNA fragments 500bp in size could be seen. I proceeded to extract the DNA represented by the bands. I used the QIAquick Gel Extraction Kit and followed the protocol supplied with the kit. However, I added 10ul of sodium acetate, 3M, pH 5.2, to the mixture of solubilised agarose and QG buffer regardless of the color of the QG buffer.
Upon the addition of isopropanol, a white precipitate formed, and appeared like the interphase observed in phenol chloroform extraction of DNA. Following the protocol, I mixed the solution well. The precipitate was thick enough to clog my pipette tips. After vigorous mixing, I proceeded with the protocol.
After elution, I checked the quality and quantity of the DNA using a Nanodrop ND1000 spectrophotometer. The OD260/280 was 25.84, and the yield was 5.4ng/ul (in 40ul of Buffer EB).
Can someone explain to me (1) what caused the poor yield and quantity (2) what the white precipitate that formed was likely to be (3) if the substance that the spectrophotometer detects is even DNA.
Thank you!
Sounds as if you have to do it again. To answer your questions more specifically, I had a similar issue. It is possible that you had a tiny amount of contamination by any of the following which can cause a precipitation in your DNA: ethanol, SDS, acetone. Alternatively, the QG buffer that you are using for you extraction is possible to be old/gone off as after one year of use, the chaotropic salts inside (guanidine, urea, etc) tend to lose homogeneity and form crystals. If you have pipetted QG from such a solution, it is possible that you got a different amount of chaotropic salt that indicated, which can precipitate in IPA quite easily and drag the DNA down with it into the precipitate, thus accounting for your disappeared DNA.
From personal experience with the Qiagen extraction kit I can recommend the following:
- cut your gel slices as close to the DNA as possible to have the least ammount of possible contaminants in your DNA slice and maximise the concentration of DNA in your sample. Also, do it quick under the UV light as it can damage your DNA significantly, especially if you want to use it for an enzyme related application afterwards such as in vitro transcription.
- don't use very old QG, or QG that has visible deposits of yellow crystals on the walls of the bottle or a thin film covering the surface. Basically check for homogeneity as it is vital for it to work proper.
- Add an extra step of washing with PE buffer (the ethanol containing one) to be 100% sure that you got rid of everything that was chaotropic in nature
- Don't use acetate unless you need it. If the thing is yellow, it means that the indicator shoes the right pH, which is always critical to the process. The yellow indicates slightly acidic with the indicator they use. If it already slightly acidic as it should be, and you add more acetate at 5.0 pH, then you lower the pH effectively taking it away form its optimal point. Do this only if it is violet (very basic, should almost never see this) or slightly orange (neutral to basic).
- Make sure all the ethanol is gone after the PE wash. I usually just let it spin for one more minute to get it dry and without any PE. This will wreak havoc in your in vitro experiments thereafter if you keep it there and will prevent the elution of DNA off the column as it acts as an antisolvent. If you are using a vacuum manifold instead of a centrifuge then keep it on the manifold for 1-2 min longer.
- For elution use MilliQ water instead and use only 30 uL. Increase the incubation time with water to 3-5 minutes. This way all the DNA bound to the column will dissociate. Also, if the MQ water you use is hot (~70 C), the yield of DNA elution tends to be higher.
To answer your final question, you can solubilise some of the stuff in MilliQ water with 2% DMSO and try to PCR amplify it again using the same primers. If it works then your DNA has gone into the precipitate. Otherwise you lost it in a truly mysterious way (column was leaking or deffective ??). You can try the same with the eluted DNA.
Let me know if it works this time and I hope this helps.
Best,
Andrei
Hi Nathanael,
If the QG buffer turned to light yellow color after incubation for 10 mins at 50oC, you no need to add the sodium acetate. I think you did not completely dissolve your gel that's why you saw white precipitate after adding the isopropanol. You may prolong the incubation time at 50oC (and remember to shake every 2 mins) to make sure the gel is completely dissolve.
You may reduce your elution buffer volume (but not less than 30uL) to make your purified products more concentrated. If it's still too dilute, you may use concentrator to concentrate back your products. You may also heat your elution buffer (usually I will put the whole elution buffer bottle into 60oC oven before I start the purification process) before you add to the column, this will help you yield better quantity of purified products.
Regards,
Eric
Hello Nathanael,
In my experience these gel extraction kits present probably the worst method for isolating DNA from agarose gel. I have done many cloning experiments during my time in the lab and by far the best approach in terms of yield and quality of recovered DNA was the use of dialysis tubing, even for small DNA fragments. 1) the yield is possibly low for you low because a significant proportion of the DNA remains on the spin column 2) the white precipitate is likely to be agarose. The low quality of the DNA you are seeing may arise for various reasons. Nicking can occur if the gel is left under UV light for too long when you cut your bands. Solutions to this could be to cut bands faster or better still, use a blue light illuminator if possible as the blue light will not cause damage to your DNA.
Hope this helps
Dr. Robert Heeley
Sounds as if you have to do it again. To answer your questions more specifically, I had a similar issue. It is possible that you had a tiny amount of contamination by any of the following which can cause a precipitation in your DNA: ethanol, SDS, acetone. Alternatively, the QG buffer that you are using for you extraction is possible to be old/gone off as after one year of use, the chaotropic salts inside (guanidine, urea, etc) tend to lose homogeneity and form crystals. If you have pipetted QG from such a solution, it is possible that you got a different amount of chaotropic salt that indicated, which can precipitate in IPA quite easily and drag the DNA down with it into the precipitate, thus accounting for your disappeared DNA.
From personal experience with the Qiagen extraction kit I can recommend the following:
- cut your gel slices as close to the DNA as possible to have the least ammount of possible contaminants in your DNA slice and maximise the concentration of DNA in your sample. Also, do it quick under the UV light as it can damage your DNA significantly, especially if you want to use it for an enzyme related application afterwards such as in vitro transcription.
- don't use very old QG, or QG that has visible deposits of yellow crystals on the walls of the bottle or a thin film covering the surface. Basically check for homogeneity as it is vital for it to work proper.
- Add an extra step of washing with PE buffer (the ethanol containing one) to be 100% sure that you got rid of everything that was chaotropic in nature
- Don't use acetate unless you need it. If the thing is yellow, it means that the indicator shoes the right pH, which is always critical to the process. The yellow indicates slightly acidic with the indicator they use. If it already slightly acidic as it should be, and you add more acetate at 5.0 pH, then you lower the pH effectively taking it away form its optimal point. Do this only if it is violet (very basic, should almost never see this) or slightly orange (neutral to basic).
- Make sure all the ethanol is gone after the PE wash. I usually just let it spin for one more minute to get it dry and without any PE. This will wreak havoc in your in vitro experiments thereafter if you keep it there and will prevent the elution of DNA off the column as it acts as an antisolvent. If you are using a vacuum manifold instead of a centrifuge then keep it on the manifold for 1-2 min longer.
- For elution use MilliQ water instead and use only 30 uL. Increase the incubation time with water to 3-5 minutes. This way all the DNA bound to the column will dissociate. Also, if the MQ water you use is hot (~70 C), the yield of DNA elution tends to be higher.
To answer your final question, you can solubilise some of the stuff in MilliQ water with 2% DMSO and try to PCR amplify it again using the same primers. If it works then your DNA has gone into the precipitate. Otherwise you lost it in a truly mysterious way (column was leaking or deffective ??). You can try the same with the eluted DNA.
Let me know if it works this time and I hope this helps.
Best,
Andrei
10 Tips For Better DNA Gel Extraction Results
1. Trim the Gel Slice as Much as Possible
2. Minimize Exposure to UV Light
3. Remove all Traces of Phenol Using a “Home Brew” Method
4. Change to a New Brand or Bottle of Agarose
5. Run Controls
6. Renature the DNA
The melting step combines high amounts of chaotropic salts with heat. This combination will denature the DNA. If the eluted DNA appears half the expected size (it is now single-stranded), re-nature the DNA by warming up to 95°C for a minute and let cool slowly to room temperature.
7. Wash it Again
An extra wash step with the ethanol containing wash buffer in the kit will always help get rid of chaotropic salt residue on the membrane. Carry over of the salts will inhibit ligase.
8. Make Sure all of the Ethanol is Gone
9. Make Sure Your Ethanol is the Good Stuff
10. Elute with Hot Elution Buffer
Heating the elution buffer to 70°C before applying it to the column will release more of the DNA from the membrane, resulting in higher yields. Allowing the buffer to sit on the column for 5 minutes before centrifugation can also help.
Dear all,
Thank you all for your valuable responses and suggestions. I have taken all of them into consideration, and attempted to re-extract the DNA from the gel. This time I did not add the sodium acetate to the QG buffer after incubation. I also mixed the QG buffer vigorously to ensure that the solution is homogenous. The white precipitate still formed after the addition of isopropanol, and the solution became very cloudy after mixing. This time, after washing the membrane with PE buffer, I noticed that there was something orange (similarly colored to QG buffer) being retained on the silica membrane. I poked on it gently with a pipette tip and found a jelly-like substance on the membrane. Perhaps it was indeed agarose. This is strange since I ensured that the solubilisation with QG buffer was complete, and that no visible agarose was left behind. The solution was completely clear and orange. Maybe the jelly-like substance was the precipitate that formed after the addition of isopropanol. I don't know if isopropanol causes agarose to form a white precipitate.
After the entire extraction procedure, my OD260/280 was very poor, at about 20. My yield was 94.8ng/ul, but I suspect that the high absorption of light at 260nm was not due to DNA.
I re-attempted to extract the DNA from the gel, this time using the NucleoSpin Gel and PCR Clean-up kit (MACHEREY-NAGEL). I followed the manufacturer's protocol. I checked the quality and quantity of the DNA in the eluate using the Nanodrop ND1000 spectrophotometer. The OD260/280 was 1.90, and the yield was 60.4ng/ul.
I guess it was really the QIAquick Gel Extraction Kit that caused my poor DNA quality and yield. Perhaps the reagents were of poor quality for that batch? It can't be the reagents expiring since they quite new.
Again, thank you all for the help and giving me further insight.
Dear Nathanael,
yes, I believe thsi kit might be off, contaminated, or something of the sort. However, your yield of extraction is not that low. For large pieces of DNA i usually get 100-200 ng/uL, while with smaller pieces I get 20-30 (just about enough for most common applications such as Gibson assembly, ligation, Quickchange PCR, etc.).
My advice to you is to get it sequenced (you need about 8 uL of solution at around 90 ng/uL of concentration for that to work just to see if it is the right stuff. If it is, then it is pure (since coming from a single band on a gel), so you can literally just amplify it using PCR once more, kill the enzymes as 95* C afterwards and then use it just like that for your further steps.
Alternatively, get a new kit and try it with that one as there seems to be something very wrong about this one. Try changing the agarose as well next time.
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