Oleg Gusev, Rajeev Brahma, Peter Wend, Reza Nemati, Arjan Groot, Debaprasad Parai, Michelle Wu, Trung Kien Nguyen, Tatiana Kapelinskaya, Simran Bhullar .. Thank you very much for all your valuable suggestions.

My impression is that the step were you heat the sample in the first buffer and vortex it is very important. In case you can you heat your sample at 50 C and shake it for 15 minutes at the same time the yield will probably improve. I also had problems with low yield. Than I used the QIACube and I basically got 100% recovery. The only difference being that the QiaCube shakes the sample for 15 minutes at 50 C. Also, sometimes chemicals from the Qiagen gel extraction kit interfere with measuring DNA concentrations.

I agree with Harrie. Incubate the first buffer longer on 50 C.

We use Qiagen gel extraction kit and we have not faced such problem with concentration of DNA. How much DNA do you load on the gel? Load more DNA on the gel and take care that you excise the gel without loss of the DNA band. After adding buffer QG, make sure that the gel slab is dissolved properly. A gel slab of 100-200 mg will dissolve completely when you incubate it at 50C for 5-10 min, with gentle shaking in between. You can elute the DNA in 30 ul of elution buffer/dH2O, and also repeat the elution step to increase the conc. Check by running the eluted DNA on agarose gel. I think you should get 90-95% recovery after extraction.

Good luck..

I agree with my colleagues and want to add that DNA of certain sizes sometimes need additional steps and handling with the kit.

Hi,

the gel extraction has difference steps. sometimes the problems back to the previous. i mean when you have no good concentration you had better check the PCR protocol specially the master mix that you used. Washing is an important step during gel recovery. the concentration of agarose should be considered.

Please have a look at the attachment file.

i hope you like it

Good Luck

Hi Krithi,

Have you check in agarose gel after you have done the extraction. Sometimes it seems a problem for nanodrop that it cannot measure properly the exact concentration of DNA either for its very low concentration (though the lower detection limit for nanodrop is 0.4 or 2 ng/microlt, depending on model) or contamination of sample with residual reagents you have used during extraction procedure. But in this case you can cross-check with your gel picture by running it in gel !! If you get clear band loading 2-3 microlt then its fine, you can go ahead then. And here is a useful link for you why you get a descending slope instead of peak. Hope it will help you. Best of luck !!

http://www.nanodrop.com/Library/T042-NanoDrop-Spectrophotometers-Nucleic-Acid-Purity-Ratios.pdf

http://www.nanodrop.com/ND1/NucleicAcid-Booklet.html

Low DNA concentration can be many reasons. It could be simply that you don't have enough DNA to start with and the DNA mini columns is not good enough to recover such a small amount of DNA, you can try Tini DNA/RNA spin columns, which is designed to recover and concentrate trace amount of DNA or RNA and the elution volume is as small as 5ul. All the buffers for Tini spin columns is compatible with the ones in Mini columns, so you only need to buy bulk Tini columns and use the solutions in Qiagen gel extraction kit. We also have gel extraction kits with both Mini or Tini columns ($55/50preps). If you want to make you own solutions in the kits, please contact [email protected] for the solution recipes in DNA miniprep, DNA gel extraction and PCR cleanup kits. Good luck!

Tini column: http://www.enzymax.net/columns/tini_spin_DNA_column.htm

Hi

I agree with Dr.Harrie. I have one more comment to share with you that you should load your DNA in the low gelling temperature agarose (Type VII-A) with EEO

What is the size of your fragment? Add 1 gel volume of isopropanol to your sample, if the size of DNA fragments 4kb, as recomended in instruction.http://www.qiagen.com/Products/Catalog/Sample-Technologies/DNA-Sample-Technologies/DNA-Cleanup/QIAquick-Gel-Extraction-Kit#resources

Hello Sai,

Tatiana Kapelinskaya rightly pointed out the use of isopropanol if the size of the fragment is below or above the range. I also faced this problem at one or the other time.

Secondly decrease the volume of elution to 30 ul and do this step twice in 2 different eppendorfs, so you have slightly concentrated first tube and in case you are loosing some DNA in the column, the second elution should recover that.

Thirdly, you can warm the elution buffer or water, whatever you are using to 50 degrees and after adding the buffer or water to the column prior to centrifuging, let it stand at room temp for 3-5 minutes and then elute. Repeat it for the second elution too.

And if you are eluting an extremely long fragment like 10 kb or so, you can go for gel electrophoresis eluion in a dialysis bag. The yield is amazing, you can recover 90% of your DNA.

Best Wishes

Oleg Gusev, Rajeev Brahma, Peter Wend, Reza Nemati, Arjan Groot, Debaprasad Parai, Michelle Wu, Trung Kien Nguyen, Tatiana Kapelinskaya, Simran Bhullar .. Thank you very much for all your valuable suggestions.

UV exposure to DNA on gel deteriorates DNA quality. that's why one should not give long exposure to UV if one intends to purify the DNA. Also, extra gel should be completely removed to get sufficient amount.

Use a less amount of elution buffer/ H2O to elute the DNA. and increase the standing time before centrifugation after adding the elution buffer.

What is the size of your DNA ? Size of the DNA matters in the retention of column material. 

I have low recovery of DNA fragments obtained by PCR and purified with HiYIELD gel/pcr DNA fragments extraction kit (RBCBioscience). My fragment size is 500bp and I eluted it with 40 ul of water....

I would not trust a UV-Vis reading for DNA fragments purified with the agarose gel purification kit, b.c. Gu thiosyanate absorbs strongly in the UV. If one reprecipitates the DNA with EtOH one can get proper spectra (the peak at 260 etc).

For increased recovery from qiagen silica columns (gel or no gel) I do binding at slow speed (2,000g), washing at slow speed (ditto), drying at max speed, elution at max speed. Warming works for large fragments (>5 kb?). For PCR prods between 500 and 1000 bp I usually get 80% back.

Normally pre-warming the buffer to 50-60 degrees and let stand for two minutes works well to increase the yield. But I agree that we always loose part of the material.