Hey guys I've been using the Qiagen gel extraction kit to purify my DNA post restriction digest. I seem get very low yield but my big problem is the purity of the DNA product. When I spec it I get quite a large peak at 230 nm. This leads to me to believe that I might have used slightly too much QC buffer and Guanidinium salts are still present. Does this sound like a likely scenario and if so can it be removed? I was thinking of using the speedyvac, dialysis or spin concentration tubes. Any help would be greatly appreciated.
Best wishes
Harry
Dear Harry,
For running the gel you should use a fresh buffer and agarose gel. Your gel running tray should be properly cleaned.
Before adding QG buffer you measure the weight of your gel slice. Then you add 3 volume of QG buffer to 1 volume of gel slice.
If your gel slice is 200 mg then you add 600 μl QG buffer. Melt the gel at 50°C and properly mix it by pipetting or vortexing then loads onto the column.
You also give an additional wash with washing buffer before eluting your DNA.
I think this will help you.
Best of luck
The most important step during purification is the extraction procedures. If is not good even you use the best kit, you cannot get it. Protein contamination may interfere during purification. I agree with viji viji.
Dont forget kit doent mean best result
First, you check the quality of your DNA before restriction digestion measuring 260:280/260:230 ratio.
After gel extraction of post restriction digest product, If your peak is at 230 nm, I think that is due to guanidium salt. An extra wash (even 2) with PE buffer can help you.
Hello, there are some good tips I have heard to reduce this carryover. Sometimes the salt buffer gets stuck on the lid of the column or in the gap between the lid and column. If small droplets remain underneath the lid or in the gap between the lid and the column and the volumes of the wash buffers are not sufficient to fill the column entirely and to reach the lid, these residues are flushed into the eluate.
You can try:
Carefully load the sample to the column and try to avoid contamination of the upper part of the column and the column lid
Apply the wash buffer to the inner rim of the column
Wash 2-3 times with wash buffer in this fashion.
If you use a smaller tip like 200uL and load smaller volumes into the center of the column it may also help avoid liquid getting trapped between lid and column.
You may want to try Macherey Nagel brand: http://www.mn-net.com/tabid/1452/default.aspx
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