Team:TU Delft/23 June 2010 content

From 2010.igem.org

(Difference between revisions)
(Media and solutions)
 
(3 intermediate revisions not shown)
Line 2: Line 2:
==Characterization of Anderson RBS sequences==
==Characterization of Anderson RBS sequences==
-
[[Team:TU_Delft/protocols/ligation|Ligations]] were performed using the overnight [http://2010.igem.org/Team:TU_Delft#/blog?blog=22_June_2010 digested BioBricks]. The following ligation reactions was performed:  
+
[[Team:TU_Delft/protocols/ligation|Ligations]] were performed using the overnight [http://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"
Line 50: Line 50:
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

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:

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!!!