Team:UT-Tokyo/Protocols

From 2010.igem.org

(Difference between revisions)
m (→New Standard: Bgl I cut)
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[[Image:Bgl1.png|200px|thumb|Using Bgl1 site to ligate BioBrick parts]]
[[Image:Bgl1.png|200px|thumb|Using Bgl1 site to ligate BioBrick parts]]
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Usually, iGEM part is digested EX cut(vector)/ES cut(insert) or SP cut(vector)/XP cut(insert).
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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.
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.
Moreover, to connect short parts, there is a problem we have to face: insert should be small fragment.
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For example, we wanted to connect pT7 promoter(46bp) to rbs(15bp).
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For example, we wanted to connect pT7 promoter (46bp) to rbs (15bp).
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To connect these parts, we tried the ligation of ES cut pT7 prometer(insert, about 60 bp) and EX cut rbs(vector).
+
To connect these parts, we tried the ligation of ES cut pT7 promoter (insert, about 60 bp) and EX cut rbs (vector).
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However, it was too thin to extract the insert
+
However, it was too thin to extract the insert.
 +
 
 +
 
 +
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, 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.
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Amp resistence
 
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To solve this problem
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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.

Revision as of 14:46, 23 October 2010

UT-Tokyo

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 promoter (insert, about 60 bp) and EX cut rbs (vector). However, it was too thin to extract the insert.


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, 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.



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