We digested bacteriphage lambda with HindIII and EcoRI, ligated the fragments into the vector pHSG398, induced E. coli to take up the vector, plated the E. coli on chloramphenicol resistant plates containing X-gal, then extracted the recombinant plasmid DNA, and finally, double-digested the recombinant plasmid with HindIII and EcoRI, and determined with fragments of the bacteriophage lambda DNA were cloned.
I am using GelAnalyzer to measure the band sizes. I have two tubes, both containing recombinant pHSG398.
One lane shows fragment sizes of 2.3kbp and 2.0kbp, the other shows fragment sizes of 3.5kbp, 2.3kbp and 2.1kbp (relative to a HindIII-lambda molecular marker).
I am having trouble understanding why the 3.5kbp fragment should be able to ligate into the plasmid.
I ran a virtual restriction digest with NEBCutter, and drew a figure summarising the results of the virtual digest, and how I picture the fragments inserting into the digested plasmid.
As far as I can see, all the fragments would be able to insert into the plasmid, except those at the left and right ends of the bacteriphage genome.
For example the fragment with HindIII overhangs on either side would ligate to a fragment with HindIII-EcoRi overhangs, and then that would ligate into the plasmid.
But according to the lecturer, and the results, only a few fragments are actually cloned, and one of mine is the 3.5kbp fragment which is the left-hand side terminal end of the bacteriophage!
Which fragments would possibly clone? And why is the terminal fragment appearing in the results? Is it just a result of a poor calculation of fragment size?