Hi everyone,
Last week, I used the kit to purify a linear plasmid after digest with PCR purification protocol, but just obtained low DNA yield (2 ug linear plasmid input and only 250 ng for output). I used nuclease free water for eluting. I have no idea what the reason is, any suggestions on this would be helpful! Thank you.
If you have any pH strips, drop some of the water you used to elute onto it. In my experience, distilled water tends to actually be fairly acidic (possibly from dissolved CO2 from the air?) DNA dissolves much more easily at a slightly alkaline pH so a lot of times attempting to elute DNA with water means it doesn't come off the column very well, and leads to low yields. If you have the elution buffer the kit probably comes with you could try using that instead. Otherwise you could try making your own out of 10 mM tris pH 8 in double distilled water, then sterilize that by autoclaving for 15-20 minutes.
If the plasmid is large, that could also be an issue. >~9 kb tends to result in somewhat poor yield in silica columns, with yield being worse and worse the higher you go. About all you can do in that case is try heating up the elution buffer and double the amount of buffer in the binding step.
If it isn't the water or plasmid size, then I'm guessing some component of the kit is expired. In my experience this tends to be the wash buffers - they usually contain a lot of ethanol, and evaporate over time which can cause some DNA loss during the washes and mess up the pH/salt concentration of the buffers.
If you have any pH strips, drop some of the water you used to elute onto it. In my experience, distilled water tends to actually be fairly acidic (possibly from dissolved CO2 from the air?) DNA dissolves much more easily at a slightly alkaline pH so a lot of times attempting to elute DNA with water means it doesn't come off the column very well, and leads to low yields. If you have the elution buffer the kit probably comes with you could try using that instead. Otherwise you could try making your own out of 10 mM tris pH 8 in double distilled water, then sterilize that by autoclaving for 15-20 minutes.
If the plasmid is large, that could also be an issue. >~9 kb tends to result in somewhat poor yield in silica columns, with yield being worse and worse the higher you go. About all you can do in that case is try heating up the elution buffer and double the amount of buffer in the binding step.
If it isn't the water or plasmid size, then I'm guessing some component of the kit is expired. In my experience this tends to be the wash buffers - they usually contain a lot of ethanol, and evaporate over time which can cause some DNA loss during the washes and mess up the pH/salt concentration of the buffers.
Thanks Alexandra for nice explanation. I'm considering the pH of water I elute.
Hello Ngan,
In addition to pH adjustment of your elution water, you can also increase incubation time on elution step (before centrifugation) or you can thoroughly follow few troubleshooting points mentioned on low DNA yield at the end section here:
https://assets.thermofisher.com/TFS-Assets/LSG/manuals/purelink_gel_extraction_pcr_combo_qrc.pdf
Good Luck
Alexander Johnson has explained well and i second the answer. In my experience, if you are using nuclease free water that has not been stored for long time and you are sure there is no contamination, then heating up may increase the yield. in addition, repeating the elution step also increase the concentration up to some extent.
Best of luck
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