I try to do subcloning of metagenomic DNA into Plasmid. The original DNA was in fosmid vector (PCC2Fos) with an insert size of nearly 40kb ( a positive clone enzymatic activity). The strategy I have used is as follows. I use sevral restriction enzymes to digested the insert (40kb) and also the g-tube covaris techniques and it did not work. i would like to try with the sonication approach. do you have an appropriate protocole to complete subcloning step and find the gene that gives activity.
Thank you very mutch.
Moussa
Phd Student
You might consider using a 4 base recognition enzyme (like Sau3A or Taq1) and do a partially digest, purifying fragments in the right size range such as 2-3 kb. And then directly clone these into your vector. That approach generates nearly random fragments similar to what you would get with sonication but is technically easier.
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