Team:UT-Tokyo/Protocols
From 2010.igem.org
Protocols
! Notice !
-You should mix reagents well
-Put in enzymes after you mixed all other reagents
-Centrifuge the tube and collect substances on the bottom before opening a reagent
New Standard: Bgl I cut
Usually, iGEM part is digested EX cut(vector)/ES cut(insert) or SP cut(vector)/XP cut(insert). However, plasmid digested by EcoRI and XbaI is hard to extract because the length of EX cut fragment, E cut fragment and X cut fragment is almost same because EcoRI site is near to XbaI site.
Moreover, to connect short parts, there is a problem we have to face: insert should be small fragment. For example, we wanted to connect pT7 promoter(46bp) to rbs(15bp). To connect these parts, we tried the ligation of ES cut pT7 prometer(insert, about 60 bp) and EX cut rbs(vector). However, it was too thin to extract the insert
Amp resistence
To solve this problem