Team:TU Delft/6 July 2010 content

From 2010.igem.org

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Purification of the digestion products using Roche's PCR Purification Kit and subsequent overnight [https://2010.igem.org/Team:TU_Delft/protocols/ligation ligation]:
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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:
 +
 
 +
[[Image:TUDelft_undigested-digested_06_July.png|500px|thumb|left|Digestion check]]
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 +
Lane description:
 +
{|style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
 +
|'''#'''
 +
|'''Description'''
 +
|'''Expected Length (bp)'''
 +
|'''Remarks'''
 +
|-
 +
|1
 +
|SmartLadder marker (5 μL)
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|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
 +
|-
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|4
 +
|‘S – J61100 - pSB1A2 – P’
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|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’
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|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 overnight [https://2010.igem.org/Team:TU_Delft/protocols/ligation ligation] yielded:
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"

Revision as of 12:53, 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 overnight ligation yielded:

# 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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