Team:TU Delft/6 July 2010 content

From 2010.igem.org

(Difference between revisions)
Line 24: Line 24:
<h4>Characterization of Anderson RBS sequences</h4>
<h4>Characterization of Anderson RBS sequences</h4>
-
Yesterday's purified PCR product of I13401 was [[digested]] using XbaI and PstI in order to obtain a back-insert. The relevant Anderson RBS Biobricks (J61100, J61101, J61107, J61117 and J61127) were digested using SpeI and PstI. Purification of the digestion products using Roche's PCR Purification Kit and subsequent overnight [https://2010.igem.org/Team:TU_Delft/protocols/ligation ligation] yielded the 5 different RBS sequences now containing the GFP reporter regions.
+
Yesterday's purified PCR product of I13401 and the Anderson RBS Biobricks (J61100, J61101, J61107, J61117 and J61127) were [[Team:TU_Delft/protocols/restriction_enzyme_digestion|digested]]:
 +
 
 +
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
 +
|'''#'''
 +
|'''Digestion reaction'''
 +
|'''Used Buffer'''
 +
|'''Needed fragment'''
 +
|-
 +
|1
 +
| μg I13401 + XbaI + PstI
 +
|Buffer 2 (BioLabs)
 +
|‘X – I13401 – P’
 +
|-
 +
|2
 +
| μg J61100 + SpeI + PstI
 +
|Buffer 2 (BioLabs)
 +
|‘S – J61100 – P’
 +
|-
 +
|3
 +
| μg J61101 + SpeI + PstI
 +
|Buffer 2 (BioLabs)
 +
|‘S – J61100 – P’
 +
|-
 +
|4
 +
|μg J61107 + SpeI + PstI
 +
|Buffer 2 (BioLabs)
 +
|‘S – J61100 – P’
 +
|-
 +
|5
 +
|μg J61117 + SpeI + PstI
 +
|Buffer 2 (BioLabs)
 +
|‘S – J61100 – P’
 +
|-
 +
|6
 +
|μg J61127 + SpeI + PstI
 +
|Buffer 2 (BioLabs)
 +
|‘S – J61100 – P’
 +
|}
 +
 
 +
Purification of the digestion products using Roche's PCR Purification Kit and subsequent overnight [https://2010.igem.org/Team:TU_Delft/protocols/ligation ligation]:
 +
 
 +
 
 +
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
 +
|'''#'''
 +
|'''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)
 +
|}
<h4>Emulsifier</h4>
<h4>Emulsifier</h4>

Revision as of 20:27, 17 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 solved 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
1 μg I13401 + XbaI + PstI Buffer 2 (BioLabs) ‘X – I13401 – P’
2 μg J61100 + SpeI + PstI Buffer 2 (BioLabs) ‘S – J61100 – P’
3 μg J61101 + SpeI + PstI Buffer 2 (BioLabs) ‘S – J61100 – P’
4 μg J61107 + SpeI + PstI Buffer 2 (BioLabs) ‘S – J61100 – P’
5 μg J61117 + SpeI + PstI Buffer 2 (BioLabs) ‘S – J61100 – P’
6 μg J61127 + SpeI + PstI Buffer 2 (BioLabs) ‘S – J61100 – 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!
Retrieved from "http://2010.igem.org/Team:TU_Delft/6_July_2010_content"