Paris/August 6
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Digestion reactions
Redigestion reactions were performed on several PCR products already digested. This was done because of owr persistant inability to clone any PCR product.
2x reaction mixes were prepared:
- XbaI+PstI digestion reactions (buffer NEB3)
- EcoRI+PstI (NEBU buffer)
- SpeI+PstI (NEB2)
| Digestion Products | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Number | Product Name | Matrix Name | Enzyme 1 | Enzyme 2 | Size | Description | vol. left for dig. | ||
| D19' | BB dig. lox66DapAsubtilis | lox66-DapAsubtilis P6' PCR product | EcoRI | PstI | ~960 | PCR product digested for cloning as a BioBrick | 20 | ||
| D21' | bw-insert lox66-DapAsubtilis | lox66-DapAsubtilis P6' PCR product | XbaI | PstI | ~960 | lox66-DapAsubtilis P6' PCR product | 25 | ||
| D27' | lox71-ftsZ bw-insert | lox71-ftsZ PCR product P7 | XbaI | PstI | lox71-ftsZ(unmutated) ready for use as a backward insert | 30 | |||
| D28 | RBS-DapAColi BB | P8 (RBS-DapAColi) | EcoRI | PstI | ~950 | Biobrick | 15 | ||
| D29 | RBS-DapAColi | P8 (RBS-DapAColi) | XbaI | PstI | ~950 | Backward insert | 13 | ||
| D30 | RBS-DapASubtilis BB | P9 (RBS-DapASubtilis) | EcoRI | PstI | ~950 | Biobrick | 14 | ||
| D31 | RBS-DapASubtilis | P9 (RBS-DapASubtilis) | XbaI | PstI | ~950 | Backward insert | 14 | ||
| D32 | Lox71-FtsA-FtsZ | P10 Lox71-FtsA-FtsZ | XbaI | PstI | ~2600 | Backward insert - EcoRI site still presents : we can't use EcoRI | 18 | ||
| D34 | lox66 | L9' lox66 | SpeI | PstI | Biobrick plasmid carrying lox66 digested as a BV | 26 | |||
| D35 | lox71 | L10' lox71 | SpeI | PstI | Biobrick plasmid carrying lox71 digested as a BV | 33 | |||
| D36 | lox71-KmR-lox66 | P11 lox71-KmR-lox66 | EcoRI | PstI | lox71-KmR-lox66 digested as a biobrick insert | 28 | |||
Also, new digestion reactions were performed:
| Digestion Products | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Number | Product Name | Matrix Name | Enzyme 1 | Enzyme 2 | Size | Description | |||
| D18' | BB dig. lox66DapAE.coli | lox66-DapAE.coli P5 (july 30) PCR product | EcoRI | PstI | ~960 | PCR product digested for cloning as a BioBrick | |||
| D20' | bw-insert lox66-DapAE.coli | lox66-DapAE.coli P5 (july 30) PCR product | XbaI | PstI | ~960 | PCR product digested for usage as a backward insert | |||
| D37 | lox71-gfp(tripart) BI | L12.1 & L12.2 miniprep products (from 30/07 ligation reaction) | XbaI | PstI | lox71_RBS-gfp-double terminator extracted as a backward insert | ||||
| D38 | BB-insert lox66-DapAE.coli | lox66-DapAE.coli in pTOPO (P5 PCR product)(clones 1+2) | EcoRI | PstI | lox66-dapAE.coli extracted from pTOPO plasmid as a BB insert | ||||
| D39 | BI-insert lox66-DapAE.coli | lox66-DapAE.coli in pTOPO (P5 PCR product)(clones 1+2) | XbaI | PstI | lox66-dapAcoli extracted for usage as a backward insert | ||||
| D40 | BB-insert lox71-KmR-lox66 | L25.1 miniprep product (from ligation rxn of 30/07 | EcoRI | PstI | lox71-KmR-lox66 construct extracted for clonong as a biobrick | ||||
PCR on colonies
Colonies are lysed in the PCR tubes during a first step (95°C, 8')
- P6 : again (1,7) + new (9,10,11,12)
- P7 : again (1) + new (9,10,11,12,13,14,15)
- P8 : again (1,3,5,7,8) + new (9,10,11)
- P10 : again (old ones 1,5,6) + new (9,10,11,12,13)
