Team:TU Delft/23 June 2010 content

From 2010.igem.org

(Difference between revisions)
(Media and solutions)
 
(11 intermediate revisions not shown)
Line 1: Line 1:
-
===Lab Work===
+
=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#page=pages/blog&blog=22_June_2010 digested BioBricks]. The following ligation reactions was performed:
-
[[Team:TU_Delft/protocols/ligation|Ligations]] were performed using the digested BioBricks obtained in the early morning. We following ligation 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"
-
|Componet
+
|'''#'''
-
|Sample
+
|'''BioBrick'''
 +
|'''Fragment 1'''
 +
|'''Fragment 2'''
 +
|'''Recipient vector'''
|-
|-
-
|I10341
+
|1
-
|3 μL
+
|
 +
|4 μL μL ‘E–J1100–S’
 +
|3 μL μL ‘X–I10341–P’
 +
|1.5 μL ‘E–linear pSB1T3–P’
|-
|-
-
|J611XX
+
|2
-
|4 μL
+
|
 +
|4 μL μL ‘E–J1101–S’
 +
|3 μL μL ‘X–I10341–P’
 +
|1.5 μL ‘E–linear pSB1T3–P’
|-
|-
-
|pSB3C5
+
|3
-
|1.5 μL
+
|
 +
|4 μL μL ‘E–J1107–S’
 +
|3 μL μL ‘X–I10341–P’
 +
|1.5 μL ‘E–linear pSB1T3–P’
|-
|-
-
|Ligation Buffer
+
|4
-
|3 μL
+
|
 +
|4 μL μL ‘E–J1117–S’
 +
|3 μL μL ‘X–I10341–P’
 +
|1.5 μL ‘E–linear pSB1T3–P’
|-
|-
-
|T4 ligase
+
|5
-
|1 μL
+
|
 +
|4 μL μL ‘E–J1127–S’
 +
|3 μL μL ‘X–I10341–P’
 +
|1.5 μL ‘E-linear pSB1T3–P’
|-
|-
-
|H20
+
|6
-
|15 μL
+
|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==
-
We adhered to the 3:1 insert:plasmid ratio when determining the volumes of DNA added.
+
By Hugo
-
The negative control for the ligation consisted of J61100 + pSB3C5. The mixtures were incubated overnight at 16 degrees.
+
{|
 +
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!!!