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 for ligation of 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: we have no other choice to use very short fragment as the insert. For example, we wanted to connect pT7 promoter (46bp) to rbs (15bp). To connect these parts, we tried the ligation of ES cut pT7 promoter (insert, about 60 bp) and EX cut rbs (vector). However, it was too thin to extract the insert from agarose gel after electrophoresis.

To solve this problem, we propose to use BglI site which is embedded in ampicillin resistance coding region. BglI site is at a distance of about 900 bp from EcoRI site about almost all vector which is embedded in ampicillin resistance, so it is easy to connect short parts. For example mentioned above, we tried the ligation of BS cut pT7 promoter (insert, about 1000 bp) and BX cut rbs (vector). We confirm the succeed of ligation by sequencing.

Since BglI site is embedded in ampicillin resistance coding region, there is one more advantage: E.coli with unexpected ligation product cannot live because ampicillin resistance cannot express.

Since iGEM propose universal vector pSB1C3 which don’t have ampicillin resistance coding region, this method may not available next year. However, we propose to arrange EcoRI site and XbaI site / SpeI site and PstI site more far to extract enzyme digestion product precisely.

• 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