Hi,
I am having some problems to clean my PCR products. I tested the Qiagen kit and 2 types of Zymo kit (DNA clean & concentrator and gel DNA recovery kit). All of them I had a bad recovery (I have lost about 90%). So, whats going on?
My product has about 80 bp. Is it too small to clean?
I need a really clean product to use in aptamer selection (SELEX).
Thank you,
Sabrina
Hello Sabrina,
In my opinion, instead of PCR, you should generate your 80 bp DNA sequence by using annealed oligos. This will give you highly pure target DNA to be used in SELEX.
Hi Sabrina,
In cleaning up a PCR product that small you are going to lose about 50% of what you start with. The DNA clean up kits are only useful for >100bp. Smaller than that and you start to take a big loss in your recovery.
I agree with Syed that annealing oligos would be a better option but if you can't do that then my advice is to start with a lot of PCR product and expect to get 50% back after clean up.
Good luck!
Hello Sabrina,
Yet another possibility is to purify the PCR product using phenol/chloroform/isoamyl alcohol followed by EtOH precipitation. Using PCI/EthOH precipitation, you wont lose as much DNA as you do with silica column -based methods. Since you are using PCR purification kit, I assume you have optimized the PCR for your desired product and getting a single discrete band. Like Tom has said, using lots of starting material is essential to compensate for inevitable DNA loss.
I agree with Sayed A Ali, anneled oligos can work better for you. Another possibility is using G25 spin columns. GE has the illustra MicroSpin G-25 Columns that can be used for DNA larger then 10bp. You should take a look at GE website or talk to you local GE sales person. I've used home made columns with G25 for tRNA purification and it worked ok. I believe the comercial version works way better.
http://www.gelifesciences.com/webapp/wcs/stores/servlet/ProductDisplay?categoryId=11770&catalogId=10101&productId=18436&storeId=11755&langId=-1
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