Team:TU Delft/23 June 2010 content

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'''LAB WORK'''
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=Lab work=
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Ligations were performed using the digested BioBricks obtained in the early morning. The ligation protocol was in accordance with the following:
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==Characterization of Anderson RBS sequences==
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{| style="color:green; background-color:#ffffcc;" cellpadding="10" cellspacing="0" border="1"
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[[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:  
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|'''-'''
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|'''uL'''
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{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
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|'''#'''
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|'''BioBrick'''
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|'''Fragment 1'''
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|'''Fragment 2'''
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|'''Recipient vector'''
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|-
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|1
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|
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|4 μL μL ‘E–J1100–S’
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|3 μL μL ‘X–I10341–P’
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|1.5 μL ‘E–linear pSB1T3–P’
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|-
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|2
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|
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|4 μL μL ‘E–J1101–S’
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|3 μL μL ‘X–I10341–P’
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|1.5 μL ‘E–linear pSB1T3–P’
|-
|-
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|I10341
 
|3
|3
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|
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|4 μL μL ‘E–J1107–S’
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|3 μL μL ‘X–I10341–P’
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|1.5 μL ‘E–linear pSB1T3–P’
|-
|-
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|J611XX
 
|4
|4
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|
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|4 μL μL ‘E–J1117–S’
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|3 μL μL ‘X–I10341–P’
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|1.5 μL ‘E–linear pSB1T3–P’
|-
|-
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|pSB3C5
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|5
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|1.5
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|
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|4 μL μL ‘E–J1127–S’
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|3 μL μL ‘X–I10341–P’
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|1.5 μL ‘E-linear pSB1T3–P’
|-
|-
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|Ligation Buffer
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|6
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|3
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|negative control
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|'''-'''
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|3 μL μL ‘X–I10341–P’
 +
|1.5 μL ‘E–linear pSB1T3–P’
 +
|}
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We adhered to the 3:1 insert:plasmid ratio when determining the volumes of DNA added.
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The mixtures were incubated overnight at 16 °C.
 +
 
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==Media and solutions==
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 +
By Hugo
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{|
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Ramon and I prepared the DNA electrophoresis loading buffer, 10 ml of this mixture are enough for thousands of samples.
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|-
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|T4 ligase
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|}
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|1 unit
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The recipe is as follows:
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{| style="color:black; background-color:white;" cellpadding="3" cellspacing="0" border="1"
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|'''Compound'''
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|'''Amount required'''
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|'''Final concentration'''
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|-
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|Bromophenol blue
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|0.025 g
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|0.25% w/v
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|-
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| Xylene Cyanol FF
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|0.025 g
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|0.25% w/v
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|-
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|Ficoll (type 400: Pharmacia)
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|1.5 g
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|15% w/v
|-
|-
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|H20
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|Water
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|15
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|As much as you need for 10 ml
|}
|}
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We adhered to the 3:1 insert:plasmid ratio when determining the volumes of DNA added.
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The negative control for the ligation consisted of J61100 + pSB3C5 and the negative control for the transformation was pure H2O. We're hoping for some nice transformants we can finally characterize tomorrow!
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'''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!!!

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