It's confusing to find extra bands of higher molecular weight in my insert sample which was PCR purified by gel extraction method. The insert bands were very clean in 0.8% agarose gel. It was followed by double digestion with NheI and HindIII enzymes at 37 degree overnight. During the second round of gel extraction after digestion, I found two bands-one equivalent to my insert size and another band higher than my insert.
Can anyone has suffered the same problem? Please give your inputs.
Thanks.
how do you cut your agarose gel for your gel extraction? one possibility is if you re-use razor that has been used to cut other DNA fragments, that may introduce contamination of DNA.
Hello,
I am not sure why you are getting higher MW band after gel purification, may its because of denaturation at the time of gel purification. In your case i can suggest that there is no need of gel purification after restriction digestion as you already have gel purified PCR fragment. Just carry out PCR clean up after restriction digestion, check on 1% agarose gel and proceed for cloning
Regards
Thank you
how do you cut your agarose gel for your gel extraction? one possibility is if you re-use razor that has been used to cut other DNA fragments, that may introduce contamination of DNA.
Hi Gaurav,
If you still have the gel purified non digested insert, run it on agarose gel with the digested one and check if the other band is also present in the gel purified non digested insert.
Hi Gaurav,
Check your insert, may be the higher band represent the undigested insert and the lower one after digestion. I think overnight digestion of the sample is long.
@Augusta Jamin- thanks for your suggestion. I have one doubt, even if i have used the same razor to cut the insert and vector, will it be possible that razor will contain such a high detectable amount of vector in the gel?
@Jacques Davy Ibaba- Ho do I get undigested vector in my insert sample? It was gel purified PCR product after digestion.
Hi Gaurav,
I am not sure what you meant by 'high' detectable amount, is it to a comparable level as your insert? Using agarose gel, you can detect at the minimal ~10 ng of DNA. So if there is contamination around such amount of DNA, it can be detectable. You may also test other components of your digestion mix, for example the water or buffer components that you use that may possibly be the source of DNA contamination. Maybe try fresh water or a different batch of buffer. Also may be use a different box of pipette tips.
Thanks Augusta for your help. I have initiated a fresh start. It's much better than remain in ambiguity through out the experiment. I was just curious about such unsual results. It might be contamination, but still I need to explore, how?
Thanks every body for sparing your precious time.
Hi Gaurav,
One reason could be contamination as mentioned by Augusta. But in your case, I believe it is due to your prolonged reaction time, which is leading to star activity (common in double digests). I would suggest you to reduce the time of digestion and or enzyme concentration to prevent star activity. Also check if you are using compatible buffer for your restriction enzymes from NEB.
Hope this helps.
Next time when you do the double digestion, also check on the gel by loading undigested insert with the digested insert. If you see extra bands on the digested product only, then it is due to extended reaction time or high enzyme conc or incompatible buffers. But if you see extra bands in both, it could be due to contamination.
I agree with few of the above. It is very likely your double digested insert contained supercoiled vector +insert that wasn't fully digested. The higher MW band is likely your linearized vector that was carried over from the comigrated band.
Hey Gaurav,
Did you get your expected result and were you able to find the possible reason? Just curious as it could help any of us in the future if we face the same problem.
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