Why does insert has two bands after RE digestion? - (Apr/04/2008 )
Hello there. Unknown inserts were given to me and I was supposed to make transformed vectors with these inserts. These inserts were randomly given from a cDNA library. The inserts were already cut with EcoRI before given to me. After making my transformation, I subjected the transformed plasmids to RE digestion with EcoRI again. Technically, I am supposed to get two bands only, the vector band,2000++bp (which I got) and the insert band on agarose gel. However, I got 2 bands (1000++ for each band) for my insert when the inserts do not have any internal EcoRI RE site since the inserts were already cut with EcoRI in the first place. Why do i got two bands for my insert? Thank you!
how long is your insert? Are you sure the vector does not have an EcoRI site as well? It's also possible that EcoRI exhibited some star activity if the reaction conditions allowed it...
the insert size is abt 700 . that is confirmed after i did sequencing of the plasmid and managed to sequence from one Ecor1 site to the next. There is no internal EcoR1 site in the sequencing so there should not be two bands for insert. the vector does not have another Eco r1 site other than at the ligation of insert sites.
just in case this star activity, could you list the formulation for your restriction digest? Less is more when using restriction enzymes. Often using too much restriction enzyme results in bad things happening.
You have sequenced the plasmid and the insert within. And there are no other EcoRI sites
Hmm... could you do a few other restriction digest? Since you have the sequence of the insert.
This is quite perplexing, if the insert has already been sequenced.