Team:TU Delft/29 July 2010 content

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=Lab work=
=Lab work=
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==Hydrocarbon Sensing==
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====Cell bank====
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A cell bank was made from E.coli Top10 cells transformed with AlkS-E0422 and AlkS-B0015. We used 2 mL of the bacterial cells to make [[Team:TU_Delft/protocols/freezing_bacterial_stocks|-80 °C stocks]].
==Alkane Degradation==
==Alkane Degradation==
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K398007A, K398008A, K398009A, K398010A, K398017A, K398018A, K398019A, B0015 were [[Team:TU_Delft/protocols/PCR|amplified]] with the universal primers G00100 and G00101. The product was put on [[Team:TU_Delft/protocols/agarose_gel|1% agarose gel]]:
K398007A, K398008A, K398009A, K398010A, K398017A, K398018A, K398019A, B0015 were [[Team:TU_Delft/protocols/PCR|amplified]] with the universal primers G00100 and G00101. The product was put on [[Team:TU_Delft/protocols/agarose_gel|1% agarose gel]]:
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[[Image:TU Delft 29072010 PCR2.jpg|400px|thumb|left|1% agarose of PCR. Gel runned at 100V for 1 hour. Of all samples 10 μL + 2 μL loadingbuffer was loaded. 5 μL was loaded of marker]]
Lane Description:
Lane Description:
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[[Image:TU Delft 29072010 PCR2.jpg|400px|thumb|left|1% agarose of PCR. Gel runned at 100V for 1 hour. Of all samples 10 μL + 2 μL loadingbuffer was loaded. 5 μL was loaded of marker]]
 
====Digestion====
====Digestion====

Latest revision as of 09:05, 13 October 2010

Contents

Lab work

Hydrocarbon Sensing

Cell bank

A cell bank was made from E.coli Top10 cells transformed with AlkS-E0422 and AlkS-B0015. We used 2 mL of the bacterial cells to make -80 °C stocks.

Alkane Degradation

PCR Amplification

K398007A, K398008A, K398009A, K398010A, K398017A, K398018A, K398019A, B0015 were amplified with the universal primers G00100 and G00101. The product was put on 1% agarose gel:

1% agarose of PCR. Gel runned at 100V for 1 hour. Of all samples 10 μL + 2 μL loadingbuffer was loaded. 5 μL was loaded of marker

Lane Description:

# Description Expected Length (bp) Primers Status
M1 SmartLadder n/a n/a n/a
1 PCR product of 007 1616 G00101 + G00101 ✓
2 PCR product of 008 473 G00101 + G00101 ✓
3 PCR product of 009 482 G00101 + G00101 ✓
4 PCR product of 010 1505 G00101 + G00101 ✓
5 PCR product of 017 1625 G00101 + G00101 ✓
6 PCR product of 018 1052 G00101 + G00101 ✓
7 PCR product of 019 1796 G00101 + G00101 ✓
8 PCR product of B0015 445 G00101 + G00101 ✓

Digestion

The PCR products were subsequently digested:

# Sample Enzyme 1 Enzyme 2 Buffer BSA Needed fragment
1 40 μL PCR product 007 EcoRI SpeI 2 (BioLabs) ✓ ‘E–007–S’
2 40 μL PCR product 008 XbaI PstI 2 (BioLabs) ✓ ‘X–008-P’
3 40 μL PCR product 009 EcoRI SpeI 2 (BioLabs) ✓ ‘E–009–S’
4 40 μL PCR product 010 XbaI PstI 2 (BioLabs) ✓ ‘X–010-P’
5 40 μL PCR product 017 EcoRI SpeI 2 (BioLabs) ✓ ‘E–009–S’
6 40 μL PCR product 018 XbaI PstI 2 (BioLabs) ✓ ‘X–018-P’
7 40 μL PCR product 019 EcoRI SpeI 2 (BioLabs) ✓ ‘E–019-S’
8 40 μL PCR product B0015 XbaI PstI 2 (BioLabs) ✓ ‘X–B0015-P’

Ligation

The digestion products were ligated overnight:

# BioBrick Fragment 1 Fragment 2 Final volume
1 K398011 4 μL ‘E–007–S’ 4 μL ‘X–008–P’ 10 μL
2 K398012 4 μL ‘E–009–S’ 4 μL ‘X–010–P’ 10 μL
3 K398020 4 μL ‘E–017–S’ 4 μL ‘X–018–P’ 10 μL
4 K398021 4 μL ‘E–019–S’ 4 μL ‘X–B0015–P’ 10 μL
5 negative control 4 μL ‘E–007–S’ 4 μL H2O 10 μL

Plasmid isolation

A plasmid isolation was done on the positive colonies of yesterday. This was done using a Qiagen Miniprep kit. We used 2 mL of the bacterial cells to make -80 °C stocks.

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