Team:TU Delft/6 July 2010 content

From 2010.igem.org

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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:
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The digestion products were purified using Roche's PCR Purifiation Kit and loaded onto a [[Team:TU_Delft/protocols/agarose_gel|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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[[Image:TUDelft_undigested-digested_06_July.png|500px|thumb|left|1 % agarose of digestion check. Gel runned 1 hour at 100 V. Of all samples 1 μL sample + 4 μL MQ + 1 μL loadingbuffer was loaded and 5 μL was loaded of marker]]
Lane description:
Lane description:
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|'''Description'''
|'''Description'''
|'''Expected Length (bp)'''
|'''Expected Length (bp)'''
-
|'''Remarks'''
+
|'''Status'''
|-
|-
-
|1
+
|M1
|SmartLadder marker (5 μL)
|SmartLadder marker (5 μL)
|n/a
|n/a
-
|
+
|n/a
|-
|-
-
|2
+
|1
-
|Undigested J61100 (1 μL + 4 μL MQ + 1 μL Loading Buffer)
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|Undigested J61100 in pSB1A2
-
|
+
|2134
-
|Circular Plasmid
+
|<font color=limegreen>&#10004;</font>
|-
|-
|3
|3
-
|Undigested J61101 (1 μL + 4 μL MQ + 1 μL Loading Buffer)
+
|Undigested J61101 in pSB1A2
-
|
+
|2134
-
|Circular Plasmid
+
|<font color=limegreen>&#10004;</font>
|-
|-
|4
|4
-
|‘S – J61100 - pSB1A2 – P’
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|J61100 + SpeI + PstI 
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|2116
+
|18, 2116
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|Visible
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|<font color=limegreen>&#10004;</font>
|-
|-
|5
|5
-
|‘S – J61101 - pSB1A2 – P’
+
|J61101 + SpeI + PstI 
-
|2116
+
|18, 2116
-
|Visible
+
|<font color=limegreen>&#10004;</font>
|-
|-
|6
|6
-
|Undigested J61107 (1 μL + 4 μL MQ + 1 μL Loading Buffer)
+
|Undigested J61107 in pSB1A2
-
|
+
|2134
-
|Circular plasmid
+
|<font color=limegreen>&#10004;</font>
|-
|-
|7
|7
-
|‘S – J61107 - pSB1A2 – P’
+
|J61107 + SpeI + PstI
-
|2116
+
|18, 2116
-
|Visible
+
|<font color=limegreen>&#10004;</font>
|-
|-
|8
|8
-
|Undigested J61117 (1 μL + 4 μL MQ + 1 μL Loading Buffer)
+
|Undigested J61117 in pSB1A2
-
|
+
|2134
-
|Circular plasmid
+
|<font color=limegreen>&#10004;</font>
|-
|-
|9
|9
-
|‘S – J61117 - pSB1A2 – P’
+
|J61117 + SpeI + PstI 
-
|2116
+
|18, 2116
-
|Visible
+
|<font color=limegreen>&#10004;</font>
|-
|-
|10
|10
-
|Undigested J61127 (1 μL + 4 μL MQ + 1 μL Loading Buffer)
+
|Undigested J61127 in pSB1A2
-
|
+
|2134
-
|Circular plasmid
+
|<font color=limegreen>&#10004;</font>
|-
|-
|11
|11
-
|‘S – J61127 - pSB1A2 – P’
+
|J61127 + SpeI + PstI
-
|2116
+
|18, 2116
-
|Visible
+
|<font color=limegreen>&#10004;</font>
|-
|-
|12
|12
-
|Undigested I13401 (1 μL + 4 μL MQ + 1 μL Loading Buffer)
+
|Undigested I13401 in pSB1A2
-
|
+
|2936
-
|Circular plasmid
+
|<font color=limegreen>&#10004;</font>
|-
|-
|13
|13
-
|‘X – I13401 – P’
+
|I13401 + XbaI + PstI
-
|883
+
|883, 2053
-
|Visible
+
|<font color=limegreen>&#10004;</font>
|-
|-
|14
|14
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|BioRad EZ Load
|BioRad EZ Load
|n/a
|n/a
-
|Visible
+
|n/a
|}
|}

Revision as of 17:45, 31 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:

# Sample Enzyme 1 Enzyme 2 Buffer BSA Needed fragment
C1 1.15 μg J61100 SpeI PstI 2 (BioLabs) ✓ ‘S–J61100 pSB1A2–P’
C2 1.68 μg J61101 SpeI PstI 2 (BioLabs) ✓ ‘S–J61101 pSB1A2–P’
C3 1.30 μg J61107 SpeI PstI 2 (BioLabs) ✓ ‘S–J61107 pSB1A2–P’
C4 0.95 μg J61117 SpeI PstI 2 (BioLabs) ✓ ‘S–J61117 pSB1A2–P’
C5 0.51 μg J61127 SpeI PstI 2 (BioLabs) ✓ ‘S–J61127 pSB1A2–P’
D1 2.59 μg I13401 XbaI PstI 2 (BioLabs) ✓ ‘X–I13401–P’
D2 2.65 μg I13401 XbaI PstI 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:

1 % agarose of digestion check. Gel runned 1 hour at 100 V. Of all samples 1 μL sample + 4 μL MQ + 1 μL loadingbuffer was loaded and 5 μL was loaded of marker

Lane description:

# Description Expected Length (bp) Status
M1 SmartLadder marker (5 μL) n/a n/a
1 Undigested J61100 in pSB1A2 2134 ✔
3 Undigested J61101 in pSB1A2 2134 ✔
4 J61100 + SpeI + PstI 18, 2116 ✔
5 J61101 + SpeI + PstI 18, 2116 ✔
6 Undigested J61107 in pSB1A2 2134 ✔
7 J61107 + SpeI + PstI 18, 2116 ✔
8 Undigested J61117 in pSB1A2 2134 ✔
9 J61117 + SpeI + PstI 18, 2116 ✔
10 Undigested J61127 in pSB1A2 2134 ✔
11 J61127 + SpeI + PstI 18, 2116 ✔
12 Undigested I13401 in pSB1A2 2936 ✔
13 I13401 + XbaI + PstI 883, 2053 ✔
14 empty empty empty
15 BioRad EZ Load n/a n/a

Followed by over night ligation:

# 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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