Hey, just a few tips I have found help increase my product yield after purification,

First you need to make sure you understand what it is you are actually doing at each step in the protocol.

- Before you do anything remember that you need to add Ethanol to the PE buffer before it is used, this helps the DNA precipitate and be able to bind to the spin column.

- make sure that when you add the PB solution, that the resulting solution is 'yellow' and not violet, this is because DNA adsorption occurs at a pH

Maybe you have a problem with pH, as far as i remember one of the buffers in the Kit should be treated with a salt solution to have a better elution efficiency.. Maybe check the manual for that... Cause if there is a wrong pH your nucleic acids cannot bind to the column

Hi, when you add water or TE buffer in the end make sure you keep for at least a minute to dissolve DNA before spinning. Cheers, Nadine

Thanks Norbert and Nadine. I will check as you said @Norbert. But with same kit I got good band for another PCR sample! Even I did repeat and used 30 ul EB/TE buffer instead of 50 ul and kept 1 min too @Nadine.

It can also happen that you put the wrong gel slice into your tube. To avoid this, i check the gel once more on the UV table once the gel slices are in the right tubes.

Hi Ahasan,

Above all comments are absolutely correct. If you are not sure about the product, you should go for PCR again with same primer pair and it will improve your product quality. All the best.

Ahasan, how long is your PCR product? For longer PCR products isopropanol is usually added to ensure good binding. I would look in the detailed procedure in the handbook.

thanks all. if still can't get good band then i need to repeat my PCR again and do purification then might be alright! but it's not easy job :( my PCR product size is about 400bp.

Try to use AMPure XP magnetic beads if you have an access to it.

Good luck.

Can you "now" purify any PCR product with the kit? Maybe something in the kit went off like the pH of a buffer. See if you can purify the PCR product that worked in the past before trying to re-purify the 400 bp PCR product.

thanks Gary. same kit works for some samples and for some samples don't! i am doing again by reducing the spin speed and also will increase the gel percentage..i used 1% gel and now 2% gel. besides, i am repeating PCR also for that samples which was unable to give band after purify. thanks

Hey, just a few tips I have found help increase my product yield after purification,

First you need to make sure you understand what it is you are actually doing at each step in the protocol.

- Before you do anything remember that you need to add Ethanol to the PE buffer before it is used, this helps the DNA precipitate and be able to bind to the spin column.

- make sure that when you add the PB solution, that the resulting solution is 'yellow' and not violet, this is because DNA adsorption occurs at a pH

I had the same problem few months ago….after all these advices mentioned above regarding purification protocol (Qiagen kit), the concentration and the volume of the PCR product were still insufficient for “difficult PCR product sequencing”. So, I did a very simple thing- ran PCR with the same DNA sample 10 times, in 10 tubes, ran them separately on agarose gel, then cut the gel and purified all 10 PCR products, than joined them together in one tube after elution and checked the concentration on agarose gel. The only problem is the number of DNA samples. If you have too many, this is time consuming and expensive. I had only 6 samples, so it was convenient.

@Vanja: In this case I would suggest designing primers for nested PCR. Otherwise the chance can be pretty high, that you amplify things you don't want to have.

I had the same problem with Qiagen kit. I had good strong band of PCR products, but very little coming out after purification (PCR-purification kit, not gel extraction,....I just want to get rid of primers, dNTPs, salts). It was frustrating since some samples will be just enough for sequencing (use only 30 ul elution vol.) but some will be too diluted and I have to do it again with several PCR reactions and combine them later. I'm sure it's not operator or expired kit problem. After all the suffering, now we change the brand to nucleospin kit...and got good yield always, no more problem. ^ ^ So, my suggestion is ....don't get stuck on the brand reputation...if it doesnt' work for you, try others.

Thanks Nipa. I am agree with you, we should change brand sometimes. Although i also managed about 350 samples by Qiagen gel extraction kit!! Thanks all.

I will be  procedding with rna probe designing.

but before that I am performing pcr to create antisense and sense products and  then purifying them. I have performed PCR and using QIAGEN PCR purification kit, I purified the pcr products (plasmid dna with gene) and measured its concentration (in all I had 4 samples for one gene 1antisense and 1 sense respectively). Also please note for each antisense and sense I had 3 pcr reactions which I pooled them in the end and then proceeded with PCR purification. The concentrations are 61.6ng/ul, 10.3ng/ul, 14.1ng/ul and 11.3/ul. I need final concentration of 1ug of each to proceed further.

Do you know how to improve the yeild or any other method to increase concentration.

Please advise. I would appreciate if anyone can tell me how to go about .

Ratish Raman: I think you should post your question separately. Cheers, Nadine

I have  but  some one  directed  me  to  this  question  so  I posted . You  can  find my  question  and  would  be  glad if  anyone  can  advise  :)

Did anybody notice that the lost of ethanol in PE washing buffer would also cause problem? In our lab, some people may leave the PE bottle un-covered for too long, so after I re-made a fresh PE buffer, the yield is much better than before.