Team:TU Delft/6 July 2010 content

From 2010.igem.org

(Difference between revisions)
(→Lab work)
Line 4: Line 4:
The DNA from Mr Gene has finally arrived, now we're ready to build some biobricks!
The DNA from Mr Gene has finally arrived, now we're ready to build some biobricks!
-
We were so excited; we immediately solved the DNA in water and performed a [[Team:TU_Delft/protocols/transformation|transformation]] with 100 ng of the DNA and plated on the LB agar containing the appropriate antibiotic.  
+
We were so excited; we immediately dissolved the DNA in water and performed a [[Team:TU_Delft/protocols/transformation|transformation]] with 100 ng of the DNA and plated on the LB agar containing the appropriate antibiotic.  
* AlkB2 (Ampicillin)
* AlkB2 (Ampicillin)
Line 32: Line 32:
|'''Needed fragment'''
|'''Needed fragment'''
|-
|-
-
|1
+
|C1
-
| μg I13401 + XbaI + PstI
+
|1.15 μg J61100 + SpeI + PstI
|Buffer 2 (BioLabs)
|Buffer 2 (BioLabs)
-
|‘X – I13401 – P’
+
|‘S – J61100 - pSB1A2 – P’
|-
|-
-
|2
+
|C2
-
| μg J61100 + SpeI + PstI
+
|1.68 μg J61101 + SpeI + PstI
|Buffer 2 (BioLabs)
|Buffer 2 (BioLabs)
-
|‘S – J61100 – P’
+
|‘S – J61101 - pSB1A2 – P’
|-
|-
-
|3
+
|C3
-
| μg J61101 + SpeI + PstI
+
|1.30 μg J61107 + SpeI + PstI
|Buffer 2 (BioLabs)
|Buffer 2 (BioLabs)
-
|‘S – J61100 – P’
+
|‘S – J61107 - pSB1A2 – P’
|-
|-
-
|4
+
|C4
-
|μg J61107 + SpeI + PstI
+
|0.95 μg J61117 + SpeI + PstI
|Buffer 2 (BioLabs)
|Buffer 2 (BioLabs)
-
|‘S – J61100 – P’
+
|‘S – J61117 - pSB1A2 – P’
|-
|-
-
|5
+
|C5
-
|μg J61117 + SpeI + PstI
+
|0.51 μg J61127 + SpeI + PstI
|Buffer 2 (BioLabs)
|Buffer 2 (BioLabs)
-
|‘S – J61100 – P’
+
|‘S – J61127 - pSB1A2 – P’
|-
|-
-
|6
+
|D1
-
|μg J61127 + SpeI + PstI
+
|2.59 μg I13401 + XbaI + PstI
|Buffer 2 (BioLabs)
|Buffer 2 (BioLabs)
-
|‘S – J61100 – P’
+
|‘X – I13401 – P’
 +
|-
 +
|D2
 +
|2.65 μg I13401 + XbaI + PstI
 +
|Buffer 2 (BioLabs)
 +
|‘X – I13401 – P’
 +
|-
|}  
|}  

Revision as of 12:20, 20 July 2010

Contents

Lab work

Ordered DNA stocks

The DNA from Mr Gene has finally arrived, now we're ready to build some biobricks!

We were so excited; we immediately dissolved the DNA in water and performed a transformation with 100 ng of the DNA and plated on the LB agar containing the appropriate antibiotic.

  • AlkB2 (Ampicillin)
  • rubA3 (Ampicillin)
  • rubA4 (Ampicillin)
  • rubB (Kanamycin)
  • ladA (Ampicillin)
  • ADH (Ampicillin)
  • ALDH (Kanamycin)
  • bbc1 (Ampicillin)
  • AlnA (Ampicillin)
  • OprG (Ampicillin)
  • PalkS1-2 (Ampicillin)
  • PalkB (Ampicillin)
  • P(CaiF) (Ampenicillin)
  • AlkS (Kanamycin)
  • PhPFD-α (Ampicillin)
  • PhPFD-β (Ampicillin)

Characterization of Anderson RBS sequences

Yesterday's purified PCR product of I13401 and the Anderson RBS Biobricks (J61100, J61101, J61107, J61117 and J61127) were digested:

# Digestion reaction Used Buffer Needed fragment
C1 1.15 μg J61100 + SpeI + PstI Buffer 2 (BioLabs) ‘S – J61100 - pSB1A2 – P’
C2 1.68 μg J61101 + SpeI + PstI Buffer 2 (BioLabs) ‘S – J61101 - pSB1A2 – P’
C3 1.30 μg J61107 + SpeI + PstI Buffer 2 (BioLabs) ‘S – J61107 - pSB1A2 – P’
C4 0.95 μg J61117 + SpeI + PstI Buffer 2 (BioLabs) ‘S – J61117 - pSB1A2 – P’
C5 0.51 μg J61127 + SpeI + PstI Buffer 2 (BioLabs) ‘S – J61127 - pSB1A2 – P’
D1 2.59 μg I13401 + XbaI + PstI Buffer 2 (BioLabs) ‘X – I13401 – P’
D2 2.65 μg I13401 + XbaI + PstI Buffer 2 (BioLabs) ‘X – I13401 – P’

Purification of the digestion products using Roche's PCR Purification Kit and subsequent overnight ligation:

# Ligation reaction
1 3 μL I10341 + 4 μL J1100 + 1.5 μL pSB1C3
2 3 μL I10341 + 4 μL J1101 + 1.5 μL pSB1C3
3 3 μL I10341 + 4 μL J1107 + 1.5 μL pSB1C3
4 3 μL I10341 + 4 μL J1117 + 1.5 μL pSB1C3
5 3 μL I10341 + 4 μL J1127 + 1.5 μL pSB1C3
6 4 μL J1100 + 1.5 μL pSB3C5 (negative control)

Emulsifier

Pieter is testing different condition for the emulsifying Assay. To determine the emulsifying activity we set up a calibration curve with SDS. He prepared the following samples:

# Hexane (mL) Tris Buffer pH 8 (mL) 10% SDS (mL)
B 1 1.1 0
100 1 1 0.1
50 1 1.05 0.05
25 1 1.075 0.025
10 1 1.09 0.01
5 1 1.095 0.005

Kampioenen!!!!

Today the Dutch team will play the semi-final of the World Cup! Hear us cheer! Stop sound

Pieter by the BBQ
We are ready for the soccer game
the Netherlands won the semi-final!
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