When I elute out DNA for cloning purpose, on measuring nano drop reading I get very low value of A 260/230 (it's like 0.04 or 0.08 and so). Even after repeated washing of column this problem persists. I think low A 260/230 value is creating problem in cloning process. Can anyone suggest me how to overcome this problem?
Dear Sir
I think the following link is useful for you
https://dna.uga.edu/wp-content/uploads/sites/51/2019/02/Note-on-the-260_280-and-260_230-Ratios.pdf
Best reagrds
Hello Dipanshu Ghosh
Lower A260/230 values will certainly cause some hindrance/inhibition during the cloning of your target gene. This is because your DNA prep. is getting contaminated with either polysaccharides, any solvents (like EtOH, Phenol), or salts like EDTA/potassium or sodium acetate. As you have suggested, repeated washings are also not helping. Since you are eluting your DNA from the column, you must be using a kit-based method. Does the kit suggest you to add isopropanol or EtOH to a wash buffer? If yes, then add a washing step with the same buffer lacking Isoprop. or EtOH. After you're done washing, give ample time for EtOH to evaporate (you may incubate the column in a 37 degrees incubator for 30-45 mins to ensure EtOH gets completely evaporated). And then elute your DNA in the desired volume of your elution buffer (pre-heat this buffer to 40-50 degrees to increase yield). In the past, this has worked well for me. Hopefully, you will obtain purer DNA with increased A260/230 values. Wish you all the Best!
If you are trying to elute DNA from agarose gel slices using column kits, such as those provided by Qiagen or Macherey Nagel, the gel slices are dissolved in a buffer containing guanidinium thiocyanate, which has a high absorbance at 230 nM. Guanidinium thiocyanate is supposed to be eliminated from the minicolumn by the ethanol-containing wash buffer prior to elution of the DNA. However, it is quite common that some gets left over and contaminates the eluted DNA. To avoid this, it is helpful to proceed with two ethanol washes before elution and to wipe the wash collecting tube and the outside of the column with a Kleenex or Kimwipe between washes. And of course, use a fresh Eppendorf tube to collect the eluted DNA. A ratio A260/A230 of 0.04 or 0.08 is indeed rather poor; with more stringent washing of the column, you should increase the ratio to 0.2-0.5.
If the above answers do not work then try binding less dna to the purification column initially. It is quite easy to overload these columns and get poor elution of the purified product. Sometimes adding less leads to purifying more product. If this fails then try a second elution with hot (70C) elution buffer.
Use less percentage of gel and mix the DNA with the solution very well before loading it into the column. After washing, centrifuge the column for 6mins at high speed in a tabletop centrifuge. Then open the mouth of the column and keep it for at least 45min to evaporate all the alcohol. Check your elution buffer also. If it turns turbid, just discard it and take a new one. Generally, in the case of cloning, we run the digested products into low % agarose, cut the pieces, and pass through one column to concentrate it. Hope it will work.
- Badges
- Science method