Team:TU Delft/30 July 2010 content

From 2010.igem.org

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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.  
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.  
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==PCR with PFX==
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==PCR==
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Per sample the following mix was used:
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*1,5 ul 10 mM dNTPs
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*5 ul Enhancer
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*5 ul 10x PFX Buffer
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*1 ul 50 mM MgSO4
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*3 ul 5 pmol/ul Forward Primer
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*3 ul 5 pmol/ul Reverse Primer
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*0.6 ul PFX Polymerase
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*1 ul DNA Template
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*29.9 ul H2O
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PCR Program:
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The [[Team:TU_Delft/protocols/PCR|PCR]] protocol was followed for the amplification of the BioBricks.
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start:
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*2 min 95 C
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*1 min 50 C
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*1 min 68 C
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repeated 25 times:
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*1 min 95 C
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*1 min 50 C
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*1 min 68 C
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end:
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*10 min 68 C
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PCR reactions
PCR reactions

Revision as of 19:47, 3 August 2010

Contents

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 Remarks
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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