Thoughts on trouble shooting: Check expiration of your kit buffers the pH can go off. Are you running TAE or TBE gels? How fast do you gel excise the bands? if too long, you can completely fragment the DNA. Is the size of your product below the qiaquick column MW cutoff? (your use of 3% gel suggests your products are tiny...?). Did you try running eluate on a gel despite spec reading to see if you did recover any product? My guess is your product is too small, you you may need to try a different method (e.g. glass slurry, DEAE cellulose, etc).

Hi,

did you check the kit’s various solutions? Some may need to have ethanol added before using them. Otherwise, warming the elution solution may increase the yield. Also, letting the melted gel and binding buffer incubate for 1-2 min on the column before spinning may help a bit, as well as letting the elution solution incubate for 1-2 min.

Cheers, Nadine

Kindly tell that what is the actual size of your PCR product, if it is higher then try to resolve it in 1% low melting Agarose gel, cut specific band and melt it properly then purify it with your protocol ...........

My products are 600 bp. I checked with Qiagen's tech support the kit I have used is more than 2 years old! That might be the problem.

Ok, please use 1% low melting agaros and if possible use new kit for purification..........

Hi , IF you use less % of agarose , your efficiency of elution will be high. Do not try to use more than 1% of Agarose if you are going to gel purify. You can load more PCR product because you will always loose from gel. Try making 200ul reaction of PCR and use all for elution or if you have one single clear band, you don´t need to purify from gel ( depending on experiment you have do with this pcr)...

I have very strong bands/signals. I will do sanger sequencing afterward. A. Some bands are of interest for the purpose of my study so I need to cut them out of the gel and purify. I will receive a fresh new kit from the company tomorrow and will try agian. However when I purify as usual (not from gel), even though I runt use only 20 ul PCR products I get very nice yield after purification.

If you do not purify from gel , then there is no loss ( 5% loss sometimes) but as you run it on gel, you cannot get 100% from gel as it diffuse ( atleast 10-15% on agarose) and then any kit is not 100% efficient to get all DNA from agarose as it has to dissolve agarose . Try to use atleast 100ul of good PCR product and then you will get sufficient for your experiment even if you cut it from gel

Promega Gel extraction is better than Qiagen in my hands as I have tried both the kits.

I doubt it was not melted well enough to bind and ultimately purified (eluted). As suggested by others I would use 1% or 1.5% gel (I used 1% gel for 500bp, worked for me), strictly follow the protocol given in the manual, spin down the additional wash buffer (contains EtOH), and elute DNA in water/buffer(let it stand 5-10min) after warming it (optional).

Many thanks for good advices. I incubated in 50 C for 15 minutes (resuspended every 2 minutes by vortexing). I will try 1,5 % gel this time.

HI Ara! I had the same issue, with nano-drop measurements varrying from 4-8 ng/ul, but the fact is that I never had a problem using the purified template. I could perform a nested PCR when I needed to or clone the purified product into expression vectors (and of course subsequently valadate that the sequence in the vector was the one I wanted). What is the downstream reaction you wish to perform? I would suggest you proceed with the template at hand and see how it works. All the best!

Op sorry just read you wanted to do Sanger..I use Exosap to purify prior to Sanger seq and it works fine. If you are interested to specifically find out what that one band is, you can try cloning the purified amplicon into a TOPO vector and then sequencing. I know that is a lot of work, but even with optimising I am not sure you can get a high concentration of DNA after gel extraction.

Try AMPure beads, I used them for purifying NGS libraries after every reaction, they also work for size selection with different salt concentration. they work very consistently and better than columns. If you were a person like me, who always worry about encountering malfunction column by chance, this is a great solution.

aro

forget about purification!!!!! If you load say 5ul of PCR reaction on the gel and you have a single band, no visible dimers on the gel, you can either:

1-clone PCR product directly. Simply add it to ligation reaction. it works, trust me.

2-sequence PCR product directly after enzymatic purification. How to purify enzymatically? Simply add about 0,5ul ExonucleaseI + 0,5ul alkaline fosphatase to your PCR product and run following programe in your cycler-37Cfor15minutes and 80C for 15 minutes and cool down. PCR product treated this way can be used as a template for sequencing reaction.

OR if there are more bands or primer dimers on the gel, simply cut off your band, wrap it into parafilm and sqeeze. Collect the fluid without any agarose using a pipette and use it for ligation reaction in your cloning protocol.

What I wrote really works and I use it. And I really really really have BAD experience with purification kits. I hope it helps.

Just pay attention about the UV light when you check your gel and cut it: the UV light really degrades the DNA.

Also the vortex can break the DNA so as much as you can avoid it!

And if you can purify directly your PCR product with the kit (and don't load it on the gel before) it's really really better.

Hope all the tips will help you.

wow many thanks for the all good advices. I do appreciate all of them.

I was also facing this problem previously, i resolved this problem by minimize gel size, and you make sour gel is completely dissolve in QG -buffer . you always use below than 200mg gel,100mg is to good for PCR product purification.

I am using 1.2% gel for pcr product purification.

3% gel...are you analysing very small pcr product/DNA fragment?

if no...it is best to use lower agarose gels...I am basically always using 0,8%gels..this way, you get a better extraction with DNA extraction kit of various brands.sometimes it help performing a second elution with 70¨C heated elution buffer.

You have to select the right extraction kit because every kit can separate a specific molecular weight only.