Team:TU Delft/30 July 2010 content

From 2010.igem.org

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|‘E–linear pSB1T3-P’
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|1 μg pSB1K3
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|EcoRI 
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|‘E–linear pSB1K3-P’
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====Ligation====
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The digestion products were [[Team:TU_Delft/protocols/ligation|ligated]] at 4 °C:
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{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
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|'''#'''
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|'''BioBrick'''
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|'''Fragment 1'''
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|'''Fragment 2'''
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|'''Final volume'''
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|1
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|K398012
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|1 μL ‘E–009 + 010–P’
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|7 μL ‘E–linear pSB1T3-P’
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|10 μL
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Latest revision as of 08:22, 7 August 2010

Contents

Lab work

Alkane Degradation

PCR Amplification

Yesterday's were ligation mixes were amplified with the primers PCR F and PCR R. The product was put on 1% agarose gel:

Lane Description:

# Description Expected Length (bp) Primers Status Remarks
1 PCR product of 007 + 008 PCR F + PCR R ✓
2 PCR product of 009 + 010 PCR F + PCR R ✓
3 PCR product of 017 + 018 PCR F + PCR R ✗
4 PCR product of 019 + B0015 PCR F + PCR R ✓
5 PCR product of 007 (negative control) PCR F + PCR R ✗ The primers also anneal without a complete ligation
M1 SmartLadder n/a n/a n/a

Digestion

The PCR products were subsequently digested:

# Sample Enzyme 1 Enzyme 2 Buffer BSA Needed fragment
1 40 μL PCR product 007 + 008 EcoRI PstI 2 (BioLabs) ✓ ‘E–007 + 008–P’
2 40 μL PCR product 009 + 010 EcoRI PstI 2 (BioLabs) ✓ ‘E–009 + 010-P’
3 40 μL PCR product 017 + 018 EcoRI PstI 2 (BioLabs) ✓ ‘E–017 + 018–P’
4 40 μL PCR product 019 + B0015 EcoRI PstI 2 (BioLabs) ✓ ‘E–019 + B0015-P’
5 1 μg pSB1T3 EcoRI PstI 3 (BioLabs) ✗ ‘E–linear pSB1T3-P’
5 1 μg pSB1K3 EcoRI PstI 3 (BioLabs) ✗ ‘E–linear pSB1K3-P’

Ligation

The digestion products were ligated at 4 °C:

# BioBrick Fragment 1 Fragment 2 Final volume
1 K398012 1 μL ‘E–009 + 010–P’ 7 μL ‘E–linear pSB1T3-P’ 10 μL

Alkane Sensing, Solvent Tolerance and Salt Tolerance

by Pieter

I tried to assemble the remaining biobricks from various sub projects. The goal is to assemble the following:

  • AlkS + E0240
  • PalkB + J06702
  • J61101 + bbc1
  • J61101 + PhPFDalpha
  • J61101 + PhPFDbeta

To achieve this J06702, bbc1, PhPFDalpha and PhPFDbeta were amplified by PCR. The J61101 was cut open with S and P after which the biobricks were inserted. AlkS was inserted in the backbone with E0240.

PCR

The PCR protocol was followed for the amplification of the BioBricks.

PCR reactions

# Description Expected lenght (bp) Primer Status
1 Marker n/a n/a n/a
2 J06702 869 G00100 + G00101 ✗
3 bbc1 703 MF + MR2 ✓
4 PhPFDalpha 528 MF + MR2 ✓
5 PhPFDbeta 430 MF + MR2 ✓

Digestion

The PCR products were subsequently digested:

# Sample Enzyme 1 Enzyme 2 Buffer BSA Needed fragment
1 2 μL AlkS EcoRI SpeI 2 (BioLabs) ✓ ‘E–AlkS–S’
2 3 μL E0240 EcoRI XbaI 2 (BioLabs) ✓ ‘X–E0240–E’
3 16 μL PalkB SpeI PstI 2 (BioLabs) ✓ ‘P–PalkB–S’
4 40 μL J06702 PCR product XbaI PstI 2 (BioLabs) ✓ ‘X–J06702–P’
5 6 μL J61101 SpeI PstI 2 (BioLabs) ✓ ‘P–J61101–S’
6 40 μL bbc1 PCR product XbaI PstI 2 (BioLabs) ✓ ‘X–J61101–P’
7 40 μL PhPFDalpha PCR product XbaI PstI 2 (BioLabs) ✓ ‘X–PhPFDalpha–P’
8 40 μL PhPFDbeta PCR product XbaI PstI 2 (BioLabs) ✓ ‘X–PhPFDalpha–P’

Ligation

The digestion products were ligated over two nights:

# BioBrick Fragment 1 Fragment 2 Final volume
1 AlkS + E0240 10 μL ‘E–AlkS–S’ 10 μL ‘X–E0240–E’ 25 μL
2 J61101 + bbc1 7 μL ‘P–J61101–S’ 1 μL ‘X–J61101–P’ 10 μL
3 J61101 + PhPFDalpha 7 μL ‘P–J61101–S’ 1 μL ‘X–PhPFDalpha–P’ 10 μL
4 J61101 + PhPFDbeta 7 μL ‘P–J61101–S’ 1 μL ‘X–PhPFDbeta–P’ 10 μL
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