We decided that we are going to confirm such ligations by performing:
-A Digestion: with XbaI and SpeI restriction enzymes, in order to release our parts from pGEM plasmid;
-A PCR: performed with the specific forward primer for our insert and with the reverse primer for pGEM plasmid (M13), in order to confirm that, once our inserts are indeed in there, they are also in the correct frame position.
Seeing that, today we performed the digestion with XbaI and SpeI for all of our minipreps samples. Digestion lasted 3 hours.
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