Paris/August 13
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| Line 113: | Line 113: | ||
|2 | |2 | ||
|control of ligation efficiency | |control of ligation efficiency | ||
| - | | | + | |~1000 |
|- style="background: #cccccc;" | |- style="background: #cccccc;" | ||
| style="background: #ccffcc;" |C2 | | style="background: #ccffcc;" |C2 | ||
| Line 121: | Line 121: | ||
|2µl | |2µl | ||
|control of D35 digestion | |control of D35 digestion | ||
| - | | | + | |5 |
|- style="background: #cccccc;" | |- style="background: #cccccc;" | ||
| style="background: #ccffcc;" |C4 | | style="background: #ccffcc;" |C4 | ||
| Line 129: | Line 129: | ||
|2µl | |2µl | ||
|control of D42 digestion | |control of D42 digestion | ||
| - | | | + | |5 |
|- style="background: #cccccc;" | |- style="background: #cccccc;" | ||
| style="background: #ccffcc;" |L26 | | style="background: #ccffcc;" |L26 | ||
| Line 137: | Line 137: | ||
|1 | |1 | ||
| Insertion of RBS-Cre behind araC/pBad | | Insertion of RBS-Cre behind araC/pBad | ||
| - | | | + | |x100 |
|- style="background: #cccccc;" | |- style="background: #cccccc;" | ||
| style="background: #ccffcc;" |L27 | | style="background: #ccffcc;" |L27 | ||
| Line 145: | Line 145: | ||
|1 | |1 | ||
| Insertion of ftsZ behind araC/pBad | | Insertion of ftsZ behind araC/pBad | ||
| - | | | + | |6 |
|- style="background: #cccccc;" | |- style="background: #cccccc;" | ||
| style="background: #ccffcc;" |L28 | | style="background: #ccffcc;" |L28 | ||
| Line 153: | Line 153: | ||
|1 | |1 | ||
| Insertion of ftsAZ behind araC/pBad | | Insertion of ftsAZ behind araC/pBad | ||
| - | | | + | |8 |
|- style="background: #cccccc;" | |- style="background: #cccccc;" | ||
| style="background: #ccffcc;" |L29 | | style="background: #ccffcc;" |L29 | ||
|D43 (MP8.1 gfp-tripart ES) | |D43 (MP8.1 gfp-tripart ES) | ||
|5 | |5 | ||
| - | |D42 (L9 pSB1A2::lox66 | + | |D42 (L9 pSB1A2::lox66 EX) |
|2 | |2 | ||
| Insertion of gfp-tripart in front of lox66 | | Insertion of gfp-tripart in front of lox66 | ||
| - | | | + | |x100 |
|- style="background: #cccccc;" | |- style="background: #cccccc;" | ||
| style="background: #ccffcc;" |L30(.1 & .2) | | style="background: #ccffcc;" |L30(.1 & .2) | ||
| Line 169: | Line 169: | ||
|1 | |1 | ||
|cloning dapAp promotor in J61002 | |cloning dapAp promotor in J61002 | ||
| - | | | + | |x100 |
|} | |} | ||
Revision as of 23:14, 16 August 2007
Digestions
| Digestion Products | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Number | Product Name | Matrix Name | Enzyme 1 | Enzyme 2 | Size | Description | Product Left | []ng/µL | Date |
| D41 | araC/pBad-J61002 plasmid BV | L1.2 (araC/pBad-J61002 plasmid) | Spe1 | Pst1 | Vector with the pBAD promoter ready for insertion of a RBS/CDS insert | August 13 | |||
| D22 | pSB1A2 | L6.1 (rbs-CRE) | EcoR1 | Pst1 | August 13 | ||||
| D33 | RBS(B0030)-CreORF BI | L6.2 (rbs-CRE) | XbaI | PstI | RBS-Cre digestion for use as a BI | August 13 | |||
| D42 | Lox66 FV | L9 | EcoRI | XbaI | pSB1A2-lox66 as a Forward vector | August 13 | |||
| D35 | Lox71 BV | L10 | SpeI | PstI | pSB1A2-lox71 as a Backward vector | August 13 | |||
| D43 | gfp-tripart FI | MP8.1 | EcoRI | SpeI | gfp-tri part (strongRBS-ORF-T) ready for use as a forward insert | August 13 | |||
digestion mix:
- 25µl plasmid
- 1µl BSA
- 10 µl NEB2
- 60µl H2O
- 2µl Enzyme1
- 2µl Enzyme2
Ligations
| Ligations | ||||||
|---|---|---|---|---|---|---|
| Number | Insert | Insert Volume (µL) | Vector | Vector Volume (µL) | Comments | Number of colonies |
| C1 | MP3.1 digested with EcoRI (D0) | 2 | control of ligation efficiency | ~1000 | ||
| C2 | D35 (L10 lox71 dig.SP) | 2µl | control of D35 digestion | 5 | ||
| C4 | D42 (L9 lox66 EX) | 2µl | control of D42 digestion | 5 | ||
| L26 | D33.2 (L6.2 RBS-Cre XP) | 5 | D41 (L1 J61002::pbad SP) | 1 | Insertion of RBS-Cre behind araC/pBad | x100 |
| L27 | D27 (ftsZ PCR XP) | 5 | D41 (L1 J61002::pbad SP) | 1 | Insertion of ftsZ behind araC/pBad | 6 |
| L28 | D32 (ftsAZ PCR XP) | 5 | D41 (L1 J61002::pbad SP) | 1 | Insertion of ftsAZ behind araC/pBad | 8 |
| L29 | D43 (MP8.1 gfp-tripart ES) | 5 | D42 (L9 pSB1A2::lox66 EX) | 2 | Insertion of gfp-tripart in front of lox66 | x100 |
| L30(.1 & .2) | dapAp [O31+O32]0.2µM | 2 | D9.1 & D9.2 (dapAp [O31+O32]0.2µM) | 1 | cloning dapAp promotor in J61002 | x100 |
Ligation mix:
- 2µl 10x buffer
- 1µl Ligase
- XµL plasmid DNA solution
- YµL insert DNA solution
- H2O qsp 20µL
Reaction in 20µl
ON @ 4°C
