September 10th
Double restriction on PCR products
The PCR products of yesterday were cut on the primers using XbaI and SpeI. Also the empty vector pSB1AT3 was cut in this way. After digestion, the three genes (atoB, idi and ispA) will be ligated seperately into the vector. XbaI and SpeI yield compatible sticky ends, so after ligation it is impossible to know the orientation of the insert in the vector. Orientation needs to be confirmed after transformation by another double restriction with XbaI and SpeI. XbaI-SpeI ligation will destroy the restriction site (by producing the biobrick scar), while XbaI-XbaI and SpeI-SpeI ligation will preserve restriction sites.
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