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!!!

@@ 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:
-Ligations were performed using the digested BioBricks obtained in the early morning. The ligation protocol was in accordance with the following:
 {| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
-|'''-'''
+|'''#'''
-|'''μL'''
+|'''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’
 |-
-|I10341
 |3
+|
+|4 μL μL ‘E–J1107–S’
+|3 μL μL ‘X–I10341–P’
+|1.5 μL ‘E–linear pSB1T3–P’
 |-
-|J611XX
 |4
+|
+|4 μL μL ‘E–J1117–S’
+|3 μL μL ‘X–I10341–P’
+|1.5 μL ‘E–linear pSB1T3–P’
 |-
-|pSB3C5
+|5
-|1.5
+|
+|4 μL μL ‘E–J1127–S’
+|3 μL μL ‘X–I10341–P’
+|1.5 μL ‘E-linear pSB1T3–P’
 |-
-|Ligation Buffer
+|6
-|3
+|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.
 |-
-|T4 ligase
+|}
-|1 unit
+The recipe is as follows:
+{| style="color:black; background-color:white;" cellpadding="3" cellspacing="0" border="1"
+|'''Compound'''
+|'''Amount required'''
+|'''Final concentration'''
 |-
-|H20
+|Bromophenol blue
-|15
+|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
 |}
-We adhered to the 3:1 insert:plasmid ratio when determining the volumes of DNA added.
+'''WARNING:''' ''THE POWDERS ARE ELECTROSTATIC. WEAR GLOVES AND CLEAN ALL THE PLACE WITH ALCOHOL (70% v/v)... SERIOUSLY, THE BLUE STUFF IS EVERYWHERE!!!''
-The negative control for the ligation consisted of J61100 + pSB3C5. The mixtures were incubated overnight at 16 degrees.

Team:TU Delft/23 June 2010 content

From 2010.igem.org

Latest revision as of 09:24, 24 August 2010

Lab work

Characterization of Anderson RBS sequences

Media and solutions