Hi, David! Maybe you should try gel purification?

Try gel purificartion

Hi, Roman is right. Here 10 tips for DNA gel extraction:

http://nucleicacids.bitesizebio.com/articles/10-tips-for-better-dna-gel-extraction-results/

All the best, Nadine

Gel purification is not efficient, at least in my case... I am loosing most of my product during it. Maybe you can use kit's which can bind only >300 bp fragments?

for example http://products.invitrogen.com/ivgn/product/K310001

I haven't tested this one but seems to be fine

Gel purification is painful and you'll lose a lot of material. I find you can sometimes even lose material using silica columns for cleanup, especially if there's not a lot of product there! My favourite is magnetic bead protocols as I've found them best for size selection with minimal sample loss. If you have lots of cleanups to do, it can be worth the cost, if only a few samples may be not so much. You can buy commerical stuff (like Ampure) or make your own, but test it first on a known DNA size.

You can adjust the ratio of the bead/PEG mix to the volume of the PCR reaction to remove the adapter dimer but keep your samples . Here's a good explainer on how to use Ampure to size select and get rid various sized fragments with magnetic beads + PEG: http://core-genomics.blogspot.com.au/2012/04/how-do-spri-beads-work.html

Thank you very much for your explanations

I think I going to try the magnetic beads

Janette, How can I make my own magnetic particles?

Consider using less of the primers in the reaction, or a touch-down PCR to reduce non-specific amplifications?

Hi David,

Unfortunately you can't make the beads, but you can make the mixture to do the cleanups which is much,much cheaper than buying the commercial Ampure, though you might need to more testing with it. I bought the beads from ThermoFischer Scientific, they are carboxylic modified beads called SeraMag. The are initially quite expensive but last for a very long time, but you might be able to get a small sample to test out. And you also need PEG-8000 and Sodium chloride. So if you have lots of cleanups to do, it is the way to go as there is a bit of an initial investment. Basically by altering the percentage of PEG-8000 in the mixture, you can isolate different fragment sizes. Here's another blog post (with a link to very good paper with the homemade recipe) discussing how to do it and a comparison: http://www.molecularecologist.com/2012/01/a-penny-for-your-method-ampure-substitute/

Oh and David Wragg's suggestion is good as well - optimising PCR conditions can go a long way in improving results!

Thank you Janette,

I going to try the SEraMag particles.

I can´t optimising PCR because I use a comercial kit for detection of mutations for PCR + NGS.

David

You can try gel purification

I agree with Janette, if you can afford then Beads are best other wise you should go for Gel elution but ready for loss.

Hi David,

If your required product size is much bigger than 150 bp fragment then i suggest you to use Agencourt AmPure XP beads. I have used them to remove any unwanted fragments, size selection and purification of PCR product. The amount of beads you need to purify your product will be mentioned in user guide.

Gudluck

I agree with janette and abhinav. We obtain good results in our lab with beads!

Try halving the concentration of enzyme. With less enzyme you will get less primer dimer.

Dear Jainik Patel · ARIBAS

Kindly check this link and refer product manual. it will take care of the primer dimer

https://www.thermofisher.com/order/catalog/product/K310002