Paris/August 6
From 2007.igem.org
< Paris
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)
