We performed an overnight double digest of pGL4.27 plasmid using XhoI and BamHI in a 37oC water bath. After running the digest on a gel along with a 2log ladder, we observed two bands: one around 2400bp and another around 4100bp. The pGL4.27 is a 6010bp plasmid and the expected bands for this digest are 3855bp and 2155bp. What could be the reasons for this? Thank you.
Have you verified that nothing has been cloned into this vector previously? Either that or it isn't quite the vector you think it is.
Only sequencing will really tell what's in the tube I'm afraid.
Agree with Michael. Either it already has an insert in the MCS, or is misidentified.
Dear Vivien,
There is one interesting observation in your case:
- Both the bands which you observed (at 2400 and 4100 bp) were EACH 245 bp longer than the expected bands (2155 and 3855 bp).
Thus, you could probably make a single nick in the pGL4.27 instead of the double digest to check for the length of the entire linear plasmid and see if the fragments add up. Agree with Michael and Daniel.
Hope this helps.
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