Team:TU Delft/23 June 2010 content
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- | + | =Lab work= | |
- | + | ==Characterization of Anderson RBS sequences== | |
- | [[Team:TU_Delft/protocols/ligation|Ligations]] were performed using the overnight [https://2010.igem.org/Team:TU_Delft#/blog | + | [[Team:TU_Delft/protocols/ligation|Ligations]] were performed using the overnight [https://2010.igem.org/Team:TU_Delft#page=pages/blog&blog=22_June_2010 digested BioBricks]. The following ligation reactions was performed: |
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1" | {| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1" | ||
|'''#''' | |'''#''' | ||
- | |''' | + | |'''BioBrick''' |
+ | |'''Fragment 1''' | ||
+ | |'''Fragment 2''' | ||
+ | |'''Recipient vector''' | ||
|- | |- | ||
|1 | |1 | ||
- | |3 μL | + | | |
+ | |4 μL μL ‘E–J1100–S’ | ||
+ | |3 μL μL ‘X–I10341–P’ | ||
+ | |1.5 μL ‘E–linear pSB1T3–P’ | ||
|- | |- | ||
|2 | |2 | ||
- | |3 μL | + | | |
+ | |4 μL μL ‘E–J1101–S’ | ||
+ | |3 μL μL ‘X–I10341–P’ | ||
+ | |1.5 μL ‘E–linear pSB1T3–P’ | ||
|- | |- | ||
|3 | |3 | ||
- | |3 μL | + | | |
+ | |4 μL μL ‘E–J1107–S’ | ||
+ | |3 μL μL ‘X–I10341–P’ | ||
+ | |1.5 μL ‘E–linear pSB1T3–P’ | ||
|- | |- | ||
|4 | |4 | ||
- | |3 μL | + | | |
+ | |4 μL μL ‘E–J1117–S’ | ||
+ | |3 μL μL ‘X–I10341–P’ | ||
+ | |1.5 μL ‘E–linear pSB1T3–P’ | ||
|- | |- | ||
|5 | |5 | ||
- | |3 μL | + | | |
+ | |4 μL μL ‘E–J1127–S’ | ||
+ | |3 μL μL ‘X–I10341–P’ | ||
+ | |1.5 μL ‘E-linear pSB1T3–P’ | ||
|- | |- | ||
|6 | |6 | ||
- | | | + | |negative control |
+ | |'''-''' | ||
+ | |3 μL μL ‘X–I10341–P’ | ||
+ | |1.5 μL ‘E–linear pSB1T3–P’ | ||
|} | |} | ||
We adhered to the 3:1 insert:plasmid ratio when determining the volumes of DNA added. | We adhered to the 3:1 insert:plasmid ratio when determining the volumes of DNA added. | ||
The mixtures were incubated overnight at 16 °C. | The mixtures were incubated overnight at 16 °C. | ||
+ | |||
+ | ==Media and solutions== | ||
+ | |||
+ | By Hugo | ||
+ | {| | ||
+ | Ramon and I prepared the DNA electrophoresis loading buffer, 10 ml of this mixture are enough for thousands of samples. | ||
+ | |- | ||
+ | |} | ||
+ | The recipe is as follows: | ||
+ | |||
+ | {| style="color:black; background-color:white;" cellpadding="3" cellspacing="0" border="1" | ||
+ | |'''Compound''' | ||
+ | |'''Amount required''' | ||
+ | |'''Final concentration''' | ||
+ | |- | ||
+ | |Bromophenol blue | ||
+ | |0.025 g | ||
+ | |0.25% w/v | ||
+ | |- | ||
+ | | Xylene Cyanol FF | ||
+ | |0.025 g | ||
+ | |0.25% w/v | ||
+ | |- | ||
+ | |Ficoll (type 400: Pharmacia) | ||
+ | |1.5 g | ||
+ | |15% w/v | ||
+ | |- | ||
+ | |Water | ||
+ | |As much as you need for 10 ml | ||
+ | |} | ||
+ | |||
+ | '''WARNING:''' ''THE POWDERS ARE ELECTROSTATIC. WEAR GLOVES AND CLEAN ALL THE PLACE WITH ALCOHOL (70% v/v)... SERIOUSLY, THE BLUE STUFF IS EVERYWHERE!!!'' |
Latest revision as of 09:24, 24 August 2010
Lab work
Characterization of Anderson RBS sequences
Ligations were performed using the overnight digested BioBricks. The following ligation reactions was performed:
# | BioBrick | Fragment 1 | Fragment 2 | Recipient vector |
1 | 4 μL μL ‘E–J1100–S’ | 3 μL μL ‘X–I10341–P’ | 1.5 μL ‘E–linear pSB1T3–P’ | |
2 | 4 μL μL ‘E–J1101–S’ | 3 μL μL ‘X–I10341–P’ | 1.5 μL ‘E–linear pSB1T3–P’ | |
3 | 4 μL μL ‘E–J1107–S’ | 3 μL μL ‘X–I10341–P’ | 1.5 μL ‘E–linear pSB1T3–P’ | |
4 | 4 μL μL ‘E–J1117–S’ | 3 μL μL ‘X–I10341–P’ | 1.5 μL ‘E–linear pSB1T3–P’ | |
5 | 4 μL μL ‘E–J1127–S’ | 3 μL μL ‘X–I10341–P’ | 1.5 μL ‘E-linear pSB1T3–P’ | |
6 | negative control | - | 3 μL μL ‘X–I10341–P’ | 1.5 μL ‘E–linear pSB1T3–P’ |
We adhered to the 3:1 insert:plasmid ratio when determining the volumes of DNA added. The mixtures were incubated overnight at 16 °C.
Media and solutions
By Hugo
The recipe is as follows:
Compound | Amount required | Final concentration |
Bromophenol blue | 0.025 g | 0.25% w/v |
Xylene Cyanol FF | 0.025 g | 0.25% w/v |
Ficoll (type 400: Pharmacia) | 1.5 g | 15% w/v |
Water | As much as you need for 10 ml |
WARNING: THE POWDERS ARE ELECTROSTATIC. WEAR GLOVES AND CLEAN ALL THE PLACE WITH ALCOHOL (70% v/v)... SERIOUSLY, THE BLUE STUFF IS EVERYWHERE!!!