Hellow,
I have a question about PCR clean up. I amplifie my gêne CDBA From vector, and I looding it in gel agarose. after that, I cut my gel bande and I did PCR clean up.
The problem is when I mesure concentration, I had lew ratio of 260-230, it will cause issue when I do ligation !
thanks
The most likely contamination causing a low OD260/230 after column purification is guanidine HCL added as a chaotrophic salt to increase absorption of the DNA onto the column. Some of this salt has not washed away. I would re purify the dna with an additional wash step or salt precipitate the dna to get rid of the salt. Too much GuHCL will have an effect on ligation if the ratio is very low.
I agree with Paul Rutland except that the high A230 is probably due to guanidine thiocyanate, which is present in the buffer used to dissolve agarose in PCR/gel purification kits. If you are using such a kit (e.g. from Qiagen or Macherey Nagel), be sure to wash the filter of the minicolumn carefully (2 washes) and to wipe the outside of the minicolumn with tissue paper between washes. Of course, use a fresh Eppendorf tube to collect the eluted DNA.
Thiocyanate has a strong absorption coefficient at 230 nm, thus traces will show in the A230/A260 ratio, but not necessarily impair ligation if the ratio is below 1-1.5
If you see a single, bright band on your gel, you don't have to do a gel extraction purification. You load a small bit of the PCR reaction on a gel to check the size, then do a PCR cleanup on the rest of your sample.
Good luck!
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