DNA Gel Purification - Freeze & Squeeze Method
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- čas přidán 29. 08. 2024
- Purifying DNA bands from agarose gels by the Freeze and Squeeze method is the fastest and simplest gel extraction procedure there is. This video shows you how to make Freeze & Squeeze filters for a few cents and how to use them to purify DNA bands from agarose gels. In minutes you will be able get your DNA out of a gel and setup your cloning reaction. For more details and a print protocol see: patharkar.com/...
Never try yet but the method look so elegant thank for sharing ❤
Thank you and good luck.
I'm running out of template DNA and I've discovered that it's degraded. This will be a perfect method for me to cut the target DNA band out of the gel, leaving behind the degraded product, and using the extracted DNA as PCR template after. Thanks!
Your are welcome.
Thank you Rahul for sharing this nice protocol with us!
You are welcome.
Cost effective and time saving method 🎀
Thanks!
Dear Dr
This is a classy example of necessity is the mother of invention. Grateful to have learnt it. I will try it out and let you know. Glad to meet you
Thank you! Good luck and all the best
thanks, you saved my major
The are welcome!
This is so helpful. Have you ever tried this method for polyacrylamide gels?
No I have never used this method on polyacrylamide gels. Electroelution is common for those gels since the pores are very small and the gel is very strong. Sorry I don't have a definitive answer on whether it could work.
Thanks for the amazing video, we have some old Freeze & Squeeze columns from Biorad. I wanted to know if I can use these columns for PCR clean up? Do you think the DNA will bind to the filter?
Biorad Freeze and Squeeze do not really work for PCR cleanup in the sense they will not remove the buffer or other proteins. They will only remove agarose. My favorite way to cleanup PCR is by phenol:chloroform:isoamyl extraction followed by precipitation. PCR cleanup kits can also work to remove buffer and proteins but the yield is sometimes lower. I hope this helps.
Hello! Thank you for the useful info!
Tell me please, can the same method be used with RNA extraction and with PAGE?
To get DNA or RNA out of a PAGE gel typically will require electroelution not a freeze and squeeze.
@@RahulPatharkar By 'electroelution on a free and squeeze' you mean instead of centrifugation to apply electroelution on the cutout, frozen fragment, or I undesrtood wrong?
Also is the same method that you describe in the video good for RNA purification?
@@ChristinaPatra-il1jl Yes, electricity to get the nucleic acid out of the gel.
Lovely video. Very useful and simply method. I was wondering whether this technique is compatible with EMSA gels?
That is a good question. I have never used the method to pull DNA out of an ESMA gel. In theory, it would probably work since freezing disrupts the agarose gel matrix. However, the proteins attached to the DNA may influence the recovery. Sorry I do not have a definitive answer.
@@RahulPatharkar I was wondering whether it is possible to extract ssDNA using this technique?
I have never tried to get ssDNA out of a gel. However, my guess is the freeze and squeeze method should work pretty similarly for recovery of ssDNA and dsDNA. If I had to get ssDNA out of a gel I would certainly give this method a try because it is so fast and easy. If you do give it a shot for ssDNA, I would love to know the outcome.
Can i do this step before DNA clean up? I want to send my sample for sequencing. And direct gel extract by using kit gave me like 10 ng/ul for PCR product.
Yes. You could use a kit or you could use the PCI method on this channel. Either will work to cleanup for sequencing.
It is very helpful thank you. but, can used for sequencing procedure?
Yes you can if you get enough DNA and the size of your fragment is not too big. In some cases you can get better sequencing if you phenol:chloroform extract the DNA.
@atharkar And if you just pricipitate with NaOAc/ethanol and wash twice with 70% ethanol? Would be enough?
hi Rahul. Thank you for this very fast and super easy protocol. Have you ever tested it to recover RNA? what is your take on RNA recovary by this method?
I have never tried to recover RNA from a gel. However, my guess is that this method would work for RNA recovery as well. Sorry I could not be of more help.
Hello Rahul.....very simple and straight forward method of DNA purification from agarose gels. Have you tried storing the recovered DNA? If yes....how long the DNA purified using this method remains stable?
I have stored digested vectors by this method at -20 degrees Celsius for more than a year while doing various clonings into that vector. Therefore, the DNA should be stable for more than a year at -20 degrees C. I hope that helps. All the best, Rahul
@@RahulPatharkar Hi Rahul.....thanks a lot....I will surely try this method....
Wish you a Happy and Prosperous Diwali.....
@@harishbharambe7880 Thank you. Happy Diwali to you too!
Wow, excellent time saving method! Just one question if I stain with sybr safe, not EtBr, is it necessary to do phenol chloroform extraction to quantify or can I just precipitate the DNA with sodium acetate?
Good question. I have not tried this with SYBR Safe. My guess is that it will stick to the DNA without phenol:chloroform extraction but I am not sure if SYBR Safe will interfere with absorbance at 260 nm. Ethidium absorbs a lot at 260 nm and SYBR Safe absorbs a lot less at 260 nm, so you might be OK without phenol:chloroform extraction. Sorry I cannot give you a definitive answer. I would love to know the answer if you ever figure this one out.
Thanks for posting a useful video. Can the recovered DNA be used for Sanger sequencing?
Yes it can be used for Sanger sequencing, however, you have to get the volume /concentration ratio right. I found it can be more foolproof if you PCI extract the freeze and squeeze DNA prior to Sanger sequencing.
Does the Phenol-Chloroform purification can also help get rid of the TBE buffer?
Sure does. You can change the buffer of your DNA to anything you like after you have your washed pellet.
@@RahulPatharkar Thank you very much!
Hi, I did exactly what is in the video but I didn´t get any DNA... Could it be that the material of the tip filters are different? I don´t know where the problem could be..
Different filter material is possible, however, I have used a number of brands successfully. One thing I noticed is that sometimes people do no realize your DNA can be quite dilute if a lot of extra non-DNA containing agarose is surrounds the DNA band. I always preferred to cut so that my eluted DNA would be as concentrated as possible.
@@RahulPatharkar thank you for your quick answer. We don´t have success with DNA gel extraction even with columns kits... We placed micrograms of DNA in the wells and obtained a few nanograms 5 ng/ul with the columns method.
I was wondering what kind of filter tips you used? Can the filter material be the problem?
I have used Fisher tips, Eppendorph tips, and other brands. They all worked. I am not sure if every brand will work.
Can we use liquid N2 to freeze gel instead of -80?
Yes.
That's amazing, thank you. I have a question; I want to do cloning after DNA purification from the gel. Is it possible to use the same method and then proceed with the plasmid? or do I have to further purify the DNA?
You can clone the DNA directly without further purification, however, you will not be able to accurately quantify your extracted DNA with a spectrophotometer due to interference of ethidium bromide.
@@RahulPatharkar Thanks a lot
Hello, thank you for video. but i have one question. why we must use TAE rather then TBE?
TAE does not inhibit most DNA modifying enzymes while TBE does, therefore, you can use your freeze and squeeze DNA directly if you use TAE without further purification. Hope this helps. Best.
Does this works for PCR CDS product? I want to make restriction of a CDS into a plasmid.
Yes, any DNA can work.
Wait, doesn't it work without the freezing too?
No, you have to freeze the gel to get the DNA out.
Hi, thanks for the video. any advice on how much PCR product should I charge on the gel. I'm amplifiying 200pb and also getting 500pb inespecific amplificates that I need to get rid off.
Thanks
In general without knowing your specific situation, you can load as much volume of PCR that fits in your gel's wells.
@@RahulPatharkar thanks 👍
Can it be done using -20 degrees deep freezer?
Yes a -20 C freezer can work, however, you may have to freeze for a little longer than 5 minutes.
The highsest peak i have after this method & nanodropping the results is at the 230 mark, is that still considered DNA? Or is it because of the presence of EtBr, Thank You
It is because of EtBr. Please see the print protocol for this method that points this out. patharkar.com/2019/02/gel-purification-freeze-squeeze
Hi, I was just wondering since you stain your gel with EtBr and use a pipette to push the liquid from the tip, won't your pipette be contaminated with EtBr? sorry, I'm just not sure because we don't have a designated pipette for EtBr. Thank you!
Virtually all of the EtBr would end up in the bottom of the tube. Trace amounts that might end up on the pipette could be wiped off with a kimwipe and 70% ethanol. If EtBr concerns you, you could just waste the DNA that remains in the tip or you could expel that liquid with a disposable transfer pipet. I hope this helps.
Can i use liquid nitrogen?
Yes, but a regular freezer will suffice.
Did you measure the O.D of the extracted DNA?
You can get a meaningful absorbance of the DNA only after you further clean the DNA up by a method like phenol:chloroform extraction because ethidium bromide in the sample will interfere with spec readings. Phenol:chloroform can remove ethidium bromide. However, the DNA generated by this method is pure enough for setting up a ligation or similar enzymatic reaction directly.
Very smart way! Two questions: is there any size limitation of the DNA to be released? Will a vector more than 10 kb work in this method? Will the recovered DNA work with the In Fusion cloning system? Thanks!
As size goes up, recovery goes down. This is also true for commercial gel extraction kits. I use this method for 10 kb vectors. Yes it will work with Fusion, however, you could combine it with a PCI extraction if you want to increase the efficiency. patharkar.com/2019/01/dna-extraction-phenol-chloroform-method I hope this helps.
Can you give me information, how ul that you use for ligation reaction?
In a 5 uL ligation I would use 1-2 uL of freeze and squeeze DNA at most. One key is to make sure your DNA in the fragment is reasonably concentrated (i.e. don't include extra gel without DNA in your extraction). I hope this helps.
Hi there Rahul!
I tried the method and recovered around 25uL liquid from the frozen gel. When I re-ran this liquid in a gel, I see no band. Is there a way to fix this?
It could be a concentration thing. Any gel you cut that has no DNA in it dilutes your DNA. Also, no gel extraction gives you a 100% recovery, so scale accordingly.
A written protocol and more details can be found at patharkar.com/2019/02/gel-purification-freeze-squeeze
is this method good for small fragment around 250 bp ?
Yes, it works very well for small fragments.
Is tae buffer eluted dna c compatible with golden gate reactions? Can the DNA be quantified in this solution?
Golden gate compatible = yes. Quantified by spec or nanodrop = no because ethidium bromide messes up the absorbance. Techniques like ethidium bromide spot plate can work for quantification. Combine this method with PCI purification for extremely pure DNA for any application (patharkar.com/2019/01/dna-extraction-phenol-chloroform-method) that can be quantified by spec or nanodrop..
@@RahulPatharkar Hi Rahul, Another question, what is the purpose of freezing?
Sir it's awesome but is that we should only keep it on -80°C Or we can keep it in 0°C
-20°C could also work but may take a little longer to freeze the gel. The gel has to be frozen. 0°C is not recommended because it would take a really long time to freeze the gel or it may not even freeze at that temperature.
I tried this, Thank you. But I have a question. When I looked at the gel trapped above the filter under ultraviolet light, I noticed that the gel is lightning and I think it's because that part of gel still has ethidium bromide, and in this case some of the DNA with ethidium bromide was still in the gel and had not come down.
Can you help me about this please?
and sorry for my English too
The release of DNA is probably not 100% by freezing and spinning but it is better than other kit methods I have tried. Commercial kit for gel extraction have notoriously low recovery. I recommend you work with the dried agarose on the top of the filter to see if it really contains DNA or not and not just visualize fluorescence.
Can I perform PCR on squeezed DNA?
Yes you can.
I liked the very helpflul video... but the guitar sound in the background was a little bit distracting
Thanks, some like the sound and some don't.
wig omg
thanks