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

Using Bgl1 site to ligate BioBrick parts

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

• Transformation

• Colony Pick Up

• Miniprep

• Restriction Enzyme digestion（Xba1/Pst1）—Once for all

• Restriction Enzyme digestion —One by one

• PCR(Pfu Ultra)

• PCR（Ex-Taq）

• PCR（KODplus）

• Colony PCR（Ex-Taq）

• Electrophoresis

• Gel DNA Recovery

• LB　broth

• Agarose Gel

• Seaquencing

• Producing Competent Cell