Team:TU Delft/30 July 2010 content

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(→Alkane Sensing, Solvent Tolerance and Salt Tolerance)
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==Digestion==
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The PCR products were subsequently [[Team:TU_Delft/protocols/restriction_enzyme_digestion|digested]]:
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{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
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|'''#'''
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|'''Sample'''
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|''' Enzyme 1'''
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|'''Enzyme 2'''
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|'''Buffer'''
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|'''BSA'''
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|'''Needed fragment'''
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|-
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|1
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|2 μL AlkS
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|EcoRI 
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|SpeI
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|2 (BioLabs)
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|✓
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|‘E–AlkS–S’
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|-
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|2
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|3 μL E0240
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|EcoRI 
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|XbaI
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|2 (BioLabs)
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|✓
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|‘X–AlkS–E’
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|-
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|3
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|16 μL PalkB
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|SpeI 
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|PstI
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|2 (BioLabs)
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|✓
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|‘P–PalkB–S’
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|-
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|4
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|40 μL J06702 PCR product
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|XbaI 
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|PstI
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|2 (BioLabs)
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|✓
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|‘X–J06702–P’
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|-
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|5
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|6 μL J61101
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|SpeI 
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|PstI
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|2 (BioLabs)
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|✓
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|‘P–J61101–S’
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|-
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|6
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|40 μL bbc1 PCR product
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|XbaI 
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|PstI
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|2 (BioLabs)
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|✓
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|‘X–J61101–P’
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|-
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|7
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|40 μL PhPFDalpha PCR product
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|XbaI 
 +
|PstI
 +
|2 (BioLabs)
 +
|✓
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|‘X–PhPFDalpha–P’
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|-
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|8
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|40 μL PhPFDbeta PCR product
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|XbaI 
 +
|PstI
 +
|2 (BioLabs)
 +
|✓
 +
|‘X–PhPFDalpha–P’
|}
|}

Revision as of 10:19, 2 August 2010

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 with PFX

Per sample the following mix was used:

  • 1,5 ul 10 mM dNTPs
  • 5 ul Enhancer
  • 5 ul 10x PFX Buffer
  • 1 ul 50 mM MgSO4
  • 3 ul 5 pmol/ul Forward Primer
  • 3 ul 5 pmol/ul Reverse Primer
  • 0.6 ul PFX Polymerase
  • 1 ul DNA Template
  • 29.9 ul H2O

PCR Program: start:

  • 2 min 95 C
  • 1 min 50 C
  • 1 min 68 C

repeated 25 times:

  • 1 min 95 C
  • 1 min 50 C
  • 1 min 68 C

end:

  • 10 min 68 C

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–AlkS–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’
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