Hi,
I used for the first time the QIAGEN Plasmid Miniprep kit to extract DNA from my plasmids. After DNA extraction I've quantified the DNA concentration and the result is a DNA around 200ng/ul with absorbance value 260/280 = 1 (very low) and 260/230 =1.8-2.0.
The curve displayed has 2 peaks instead just one. I would like to know if it happens normally or if I have contaminated my DNA during the extraction!! I need to sequence my construct, do you mind this contamination will affect the sequencing??
Thank you all :)
Hi Ilaria,
200ng/ul is a good yield for the QIAGEN miniprep kit. However, your 260/280 ratio suggests large amount of protein contamination in your sample. I would suggest ensuring you run the prep again, but pay partcilular attention to the washing phases. I would also recommend increasing the elution time i.e. the amount of time your elution buffer is in contact with the column membrane, I recommend around 5 minutes.
Hi Ilaria,
200ng/ul is a good yield for the QIAGEN miniprep kit. However, your 260/280 ratio suggests large amount of protein contamination in your sample. I would suggest ensuring you run the prep again, but pay partcilular attention to the washing phases. I would also recommend increasing the elution time i.e. the amount of time your elution buffer is in contact with the column membrane, I recommend around 5 minutes.
- I think Colman is spot on
- Your very high 260/230 ratio (> 1.0) suggests strong salt contamination and your second peak is likely to approximate to 230nM and correspond to guanidinium in your denaturation and column pre wash buffer
- If you have a second such peak that amount of guanidinium will denature any down stream enzymes like RT or Taq
- Thus, purify your sample again and do what Colman has suggested: deliberatley wait 5 minutes after applying you ethanol based wash buffer; spin THEN REPEAT: The ethanol in the buffer deslats your column and the initial contact time facilutates the alcohol getting into the matrix which renders desalting more effective
- If having done the above you continue to see this for odd samples - which I have - purify x 2 and in my experience that will solve the problem
- Alternatively, take your eluate add 1 volume of isopropanol; spin for 20-30 minutes and wash the resulting DNA pellet by vortexing with room temp 70% ethanol x 2; Then spin for 5 min, remove supernatant; air dry for 15 min and resuspend
- This will also remove salt reducing your 260/230 ratio to < 1.0 and correspondingly eliminating that second peak
I also agree with the above, this kit usually works very reliably for me so I would check you are using the correct reagents as indicated in the protocol and also be careful with technique to avoid carry over of eluate between steps.
@ Giorgia Pertile I run the Plasmid DNA on agrose but my band is so light, what should I do?
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