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Team:Stockholm/16 July 2010
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(New page: {{Stockholm/Top2}} ==Andreas== ===LMWP ligation=== Nina back from vacation and we decided to start working harder on making our CPPs. We divided TAT, Transportan10 and LMWP between us; I...) |
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'''Procedures''' | '''Procedures''' | ||
| - | #Denaturation: 95°C, 10 min | + | #'''Denaturation:''' 95°C, 10 min |
| + | #Temperature slowly lowered to 65°C | ||
| + | #'''Annealing:''' 65°C, 5 min | ||
| + | #Temperature slowly lowered to RT (≈27°C) | ||
| + | #'''Ligation:''' Quick Ligase added | ||
| + | #*25°C, 10 min | ||
| + | #'''Transformation''': 6 μl ligation mix into Top10. Cells grown on LB agar + 25 μg/ml Cm. | ||
Revision as of 19:29, 29 July 2010
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Andreas
LMWP ligation
Nina back from vacation and we decided to start working harder on making our CPPs. We divided TAT, Transportan10 and LMWP between us; I got LMWP.
LMWP primer dilutions
All LMWP primers were diluted 100x and 1000x, to yield concentrations of ≈1.0 and 0.1 μM, respectively:
| Primer | Original conc. | 100x dil. conc. | 1000x dil. conc. |
| LMWP_C_F1 | 109 mM | 1.09 mM | 0.11 mM |
| LMWP_N_F1 | 141 mM | 1.41 mM | 0.14 mM |
| LMWP_CN_R1 | 131 mM | 1.31 mM | 0.13 mM |
| LMWP_CN_F2 | 120 mM | 1.20 mM | 0.12 mM |
| LMWP_C_R2 | 119 mM | 1.19 mM | 0.12 mM |
| LMWP_N_R2 | 139 mM | 1.39 mM | 0.14 mM |
Flanking primers are designed with EcoRI and PstI overhangs, allowing me to use the linearized pSB1C3 vector for the ligation reaction.
Ligation
LMWP designed in two versions:
- C-LMWP containing the standard Freiburg prefix.
- N-LMWP containing the Freiburg N-part prefix.
Two ligation reactions performed for each LMWP version: (1) with high concentration primers (100x dil.) and (2) with low concentration primers (1000x dil.).
Ligation mixes
| Ingredient | C-LMWP (high conc.) [μl] | N-LMWP (high conc.) [μl] | C-LMWP (low conc.) [μl] | N-LMWP (low conc.) [μl] |
| pSB1C3 vector (25 ng/μl) | 2 | 2 | 2 | 2 |
| LMWP_C_F1 | 1 | 0 | 1 | 0 |
| LMWP_CN_R1 | 1 | 1 | 1 | 1 |
| LMWP_CN_F2 | 1 | 1 | 1 | 1 |
| LMWP_C_R2 | 1 | 0 | 1 | 0 |
| LMWP_N_R2 | 0 | 1 | 0 | 1 |
| 2X Quick Ligase buffer | 10 | 10 | 10 | 10 |
| dH2O | 3 | 3 | 3 | 3 |
| Quick Ligase (added later) | 1 | 1 | 1 | 1 |
| Total volume | 20 | 20 | 20 | 20 |
Procedures
- Denaturation: 95°C, 10 min
- Temperature slowly lowered to 65°C
- Annealing: 65°C, 5 min
- Temperature slowly lowered to RT (≈27°C)
- Ligation: Quick Ligase added
- 25°C, 10 min
- Transformation: 6 μl ligation mix into Top10. Cells grown on LB agar + 25 μg/ml Cm.
