Team:TU Delft/6 July 2010 content

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Subsequent overnight [https://2010.igem.org/Team:TU_Delft/protocols/ligation ligation] yielded:
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Subsequent over night [https://2010.igem.org/Team:TU_Delft/protocols/ligation ligation] yielded:
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Revision as of 13:30, 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’

The digestion products were purified using Roche's PCR Purifiation Kit and loaded onto a 1% agarose gel for comparison with the non-digested BioBricks:

Digestion check

Lane description:

# Description Expected Length (bp) Remarks
1 SmartLadder marker (5 μL) n/a
2 Undigested J61100 (1 μL + 4 μL MQ + 1 μL Loading Buffer) Circular Plasmid
3 Undigested J61101 (1 μL + 4 μL MQ + 1 μL Loading Buffer) Circular Plasmid
4 ‘S – J61100 - pSB1A2 – P’ 2116 Visible
5 ‘S – J61101 - pSB1A2 – P’ 2116 Visible
6 Undigested J61107 (1 μL + 4 μL MQ + 1 μL Loading Buffer) Circular plasmid
7 ‘S – J61107 - pSB1A2 – P’ 2116 Visible
8 Undigested J61117 (1 μL + 4 μL MQ + 1 μL Loading Buffer) Circular plasmid
9 ‘S – J61117 - pSB1A2 – P’ 2116 Visible
10 Undigested J61127 (1 μL + 4 μL MQ + 1 μL Loading Buffer) Circular plasmid
11 ‘S – J61127 - pSB1A2 – P’ 2116 Visible
12 Undigested I13401 (1 μL + 4 μL MQ + 1 μL Loading Buffer) Circular plasmid
13 ‘X – I13401 – P’ 883 Visible
14 empty empty empty
15 BioRad EZ Load n/a Visible

Subsequent over night ligation yielded:

# BioBrick Recipient plasmid Fragment insert Final volume
1 K398500A 130 μg ‘S – J61100 - pSB1A2 – P’ 154 μg ‘X-I13401-P’ 26 μL
2 K398501A 206 μg ‘S – J61101 - pSB1A2 – P’ 247 μg ‘X-I13401-P’ 25 μL
3 K398502A 196 μg ‘S – J61107 - pSB1A2 – P’ 247 μg ‘X-I13401-P’ 25 μL
4 K398503A 201 μg ‘S – J61117 - pSB1A2 – P’ 247 μg ‘X-I13401-P’ 25 μL
5 K398504A 202 μg ‘S – J61127 - pSB1A2 – P’ 247 μg ‘X-I13401-P’ 25.5 μL
6 Ligase Control (J61117 - pSB1A2) 123 μg ‘S – J61117 - pSB1A2 – P’ None 15 μL

To all samples one-tenth of the total volume ligase buffer was added.

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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