First, agarose or acrylamide?

Second, is there a way to skip gel purification, or is it absolutely necessary?

I've usually used Qiagen for agarose extraction with good results (I often turn my gel slice sideways to remove excess agarose after cutting it out of the gel).

I usually use crush n soak for acrylamide. I have used electroelution for acrylamide once or twice - advantage is that it isn't an eq process so you can get all your DNA out. Disadvantages are you are stuck in a gel buffer (like TBE) with lots of salt, and if you have a technical error you lose everything.

Is your yield determined from UV-Vis, estimate from agarose gel, or it's viability for downstream purposes? If you need more intact happier DNA, you can use something like SYBR safe and a visi-blue light source or converter plate so you get less damage from UV and you don't have an intercalator sticking around.

Hello Guy,

Actually I think this is a very good question. You will probably get about a hundred different answers, many from people who have experienced the same problem. Like you, our lab has tested various methods for recovering DNA from agarose gels. We have worked with Qiagen. Years ago, there was a simple filter where the DNA gel band was collected into a small vial by electrophoresis. It worked well. But I don't think it is available any longer.

You could look into the Millipore gel extraction method. The gel is compressed by centrifugation and the DNA is released into a collection tube. I would be interested to hear if anyone else has used this system successfully.

Hi Guy,

I had trouble with this, I was trying to extract a range of fragments from an agarose gel and tried Qiagen and Gene Clean kits with really poor consistency of recovery. (Although for single band extraction such as plasmid we use Qiagen with fairly decent results.)

I am now using Elutrap Electroelution from Watman. You need a piece of kit (luckily someone in the department had one already) but it's worked out much better for me. Don't know if that might help?

Are you purifying fragments or whole plasmids? How much are you loading or expecting per band?

Be careful with the spin columns from the purification kits. If you overload them with DNA, you will get nothing back. This was an issue for us with miniprep kits from Qiagen and Epoch using circlegrow medium to grow the bugs/plasmids. We had to switch back to LB medium. I've had no problems with Qiagen kits for pcr cleanup or gel purification. Make sure you wash the column a few extra times with the gel purification kit to get rid of the guanidinium salts.

Hi Guy

Try GeneElute column from SIGMA ...this is the simplest column based elution approach (one type claims removal of EtBr also from the gel)..you may have to purify it further for certain downstream reactions..

bye

Hi, QIAgen kit suggests to use a minimum of 50 ul of buffer for final elution. This may not be suitable for very low quantity. You may reduce the elution volume so as to get a visible band. Yield of DNA can be increased by loading a maximum DNA in gel and excise off the excess agarose outside your band of interest using UV. I hope the kit you use works well for other samples...

Hi Guy,

I got fed up with the low yields from Qiagen a long time ago. I think they stopped doing quality analysis as their name sells so well. I even once showed my comparison between their kit and a competitor to a rep. She wanted to look into the matter, even copied that page of my labbook but never bothered to get back to me. So I am since using Invitrogen's PureLink PCR Gel Extraction Combo. The price is about the same as Quiagen, but you'll be amazed by the reproducibility. And no, I don't get paid by Invitrogen to write this... get a free sample and try it out. You'll love it...

Cheers

Whenever you're trying to get better yield of DNA from the purification process; you must follow thorough instructions of the using kit. One more point, you need to get added to your protocol that you should add equal volume of ice-cold IPA (Iso-propyl alcohol) to tour sample DNA. The use IPA helps the cross-linking between the DNA with column matrix. Then followed by the addition of Binding buffer to it. Follow this protocol, you'll definitely get better results of purified DNA than that of previous.

Best luck...

Hi!

As Doug Bintzler mentioned probably you get 100 answers and Abhshek told follow the instructions carefully

Before going to suggest, what do you exactly want to do with the elute?

If it is for seq.ing hardly you need pico grams so you need not even go for any kit. If you carefully handle things, like cut the gel piece put in an eppendorp add 10-50 uL milliQ water squeeze it spin at high speed carefully collect the DNA dissolved in milliQ and do what ever you want to do

I suggest Geneclean kit, it works fine and simple to carry on, I used many times for labeling and other purposes.

http://kirschner.med.harvard.edu/files/protocols/MPBIO_geneclean2.pdf

go to page no.9 you get information

Previously two guys asked same questions go to the links in case if you want to refer

https://www.researchgate.net/post/DNA_extraction_from_gel

https://www.researchgate.net/post/Genomic_DNA_PCR_amplification_problem

Cheers!!!

HI,

I used low melting agarose (But people in my lab also used normal agarose) with Qiagen gel extraction kit, and it works for us.. you just modify your agarose and check once again, hope it will work.

Hi Guy. I agree with Ancy. I elute in 30uL instead of the recommended 50uL. I also spin my columns three times after the EtOH step just to really get rid of all the EtOH that could have been left on the filter which my mess up my DNA. I hope that helps.

I was going to suggest the two above points that Sarah Chow made. Definitely elute in 30uL and leave whatever you elute in on the column for a good five-ten minutes before centrifugation.

Hi I agree with a few people here. Qiagen quality went down. I have not used them in years. Their kit was not reproducible. I tried a sample from zymo. Thier kits works better for me. Plus the elution volume is 6-10ul so you can get a more concentrated sample. I usually elute with 13ul to make sure I get it all. I like to elute in dnase/rnase free water. Kit is half the price of qiagen too. good luck

Hi, You can try Biomiga's Gel Extraction Kit , offered by GeniCity.com, you may ask for a sample kit at [email protected]. The recovery rate is around 90%.

Hi, Guy,

I'm facing the same problem here... Have you solved it over there?

Best regards,

Andreia

I've been frustrated recently by gel extractions as well. I recently compared BioRad's Freeze n Squeeze to QIAquick gel extraction. In both cases I got about 25% yield. These two kits use entirely different methods and, since I got identical yields, I am now wondering if something I am doing upstream is causing the low yields (e.g. could overloading a well with more than ~100 ng of DNA cause it to have a bad peak shape resulting in only ~25% of the DNA being excised with the rest being a hypothetical invisible smear?). If any one can speak to this possibility I would appreciate it.

For background: I divided a single digest (~2 ug) into two wells in a ~0.8% TAE agarose gel, gel excised and purified the two samples in parallel with the two different methods. For the gel extraction kit I loaded the dissolved slice + IPA as normal and re-loaded the flow through a second time (extra binding), did all the recommended washes and let the PE wash sit 5 minutes (get rid of all the salts), then eluted in 30 ul of 65 C EB and eluted a second time with 22 ul 65 C EB, both elutions with a 5 minute incubation before spinning. This is the protocol that gave me 25% yield. As a comparison, I further cleaned up the freeze and squeeze using the QIAquick PCR kit (with re-loading and sequential hot elution) and got ~78% yield (much better than the gel kit).

Is this thread still going? Has anyone tried crush in soaking in Guanidine HCl-based buffers, like Qiagen's PB? The advantage could be that one would use the eluate straight as input for a silica spin column.

My 2c: DNA extracted from agarose gels using the qiagen's 'yellow buffer' (Gu thiocyanate-based) is no longer a substrate for in vitro transcription by RNA pol II and mitochondrial RNAP. Agarase-digested agarose slabs do not yield good-activity DNA templates. Acrylamide-based 'crush and soak' produces good, active, clean DNA fragments that are good substrates for in vitro transcription. The downside of acrylamide is that it's more laborious, of course.

Guanidine-based buffers, e.g. Qiagene's buffer PB seems fine for DNA as a transcription tempalte. This is also consistent with this paper:

FEBS Letters

Volume 62, Issue 1, 1 February 1976, Pages 81-84

4 M guanidine hydrochloride applied to the isolation of DNA from different sources

So, guanidine chloride and guanidine thiocyanate, even though they are bunched together as 'chaotropic agents', are differnet in how they act on DNA: GuHcl is much milder than Gu SCN

Hi Guy, all:

Actually I tried ethanol precipitation method (manual method) for raw DNA and PCR product, which is cheap, give a purity similar to most of kits and yielded abou 5 folds more than most of kits. It also useful when you want to dry your sample. I hope this will help.