Team:TU Delft/29 July 2010 content

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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====

Revision as of 08:51, 13 August 2010

Contents

Lab work

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:

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 ✓
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

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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