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-	==Lab Work==	+	=Lab work=
-	<h4>Characterization of Anderson RBS sequences</h4>	+	==Characterization of Anderson RBS sequences==
-	[[Team:TU_Delft/protocols/ligation|Ligations]] were performed using the overnight digested BioBricks. The following ligation reactions was performed:	+	[[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:
-	1. I10341 + J1100 + pSB3C5	+
-	2. I10341 + J1101 + pSB3C5	+
-	3. I10341 + J1107 + pSB3C5	+
-	4. I10341 + J1117 + pSB3C5	+
-	5. I10341 + J1127 + pSB3C5	+
-	6. J1100 + pSB3C5 (negative control)	+
-	The general composition was:
	{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"		{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
-	|'''Component'''	+	|'''#'''
-	|'''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.		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!!!