Hello enveryone. Im doing a DNA digestion for clonning with 2 restriction enzymes (Bgl2 and hind 3). I diggested 15ug DNA in a 1% agarose gel but when i extract the DNA from the gel i cant get a Good A260/230 ratio. Im using a New kit from invitrogen (pcr cleanup and gel extraction kit). I have done all the recomendations of the web site but it still doesnt work. Can anyone help? I extract 128ng/uL in 50 ul
Hi Carlos, a low A260/230 ratio after gel extraction is not uncommon: in our lab we have been trying to address this issue because the final product does not seem to conform to what the manufacturers of these kits specify. However, just so you know: even if your A260/230 is low, the DNA can be used for subsequent cloning steps without problems (ligation, Gibson-type assembly and so on).
Your DNA concentration is also much higher than what we usually get with the kit that we use (Qiagen, Zymo Research). The high A230 is coming from residual Guanidine HCl or Isothiocyanate (depending on the kit) that is used to denature and extract the DNA from your gel slab. To try and get rid of it more thoroughly, I suggest you try the following: when you are doing the wash step with buffer containing EtOH (W1 for the Invitrogen kit), add the buffer to the column and let it sit for ~1min, then spin down/apply vacuum to pass it through the column. I would repeat this step one more time, and this might bring down the A230 in your final product. Best of luck!
FYI, here you can see examples of how common contaminants from buffers used in these kits influence the spectrum of DNA:
https://assets.thermofisher.com/TFS-Assets/CAD/Product-Bulletins/TN52646-E-0215M-NucleicAcid.pdf
I agree with Alessio's comment: the high A230 is due to the presence of residual guanidinium isothiocyanate (guanidinium chloride doesn't absorb, hence the problem is not visible with plasmid extraction kits). To reduce the problem, you can improve the wash step with the EtOH-containing buffer by 1) letting the wash buffer penetrate partially into the filter by giving a very short spin, then letting it sit for 30 s-1 min before spinning a second time at full speed for 1 min; 2) wiping the outside of the minicolumn and the inside of the wash collecting tube with a Kleenex or Kimwipe between washes; 3) wash a second time with the EtOH buffer, and 4) don't forget to use a fresh tube to collect the eluted DNA.
Check https://www.reddit.com/r/labrats/comments/bjro3p/abnormally_low_260230_for_qiagen_gel_extraction/
Also check https://assets.thermofisher.com/TFS-Assets/CAD/Product-Bulletins/TN52646-E-0215M-NucleicAcid.pdf
- Badges
- Science topic
- Similar topics
- Phytochemistry
- Extracts