Team:TU Delft/12 August 2010 content

From 2010.igem.org

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(New page: {| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1" |'''#''' |'''Sample''' |'''Enzyme 1''' |'''Enzyme 2''' |'''Enzyme 3''' |'''Buffer''' |'''BSA''' |...)
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=Alkane degradation=
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BioBricks number 014K and 020A (already tries before but failed) are up today. We will do a digestion and a ligation followed by a transformation in order to make these BioBricks. Next to these two BioBricks we will also be working on placing some of the previously made BioBricks in the 1C3 plasmid so we can ship it to the registry.
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==Digestion==
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
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|AseI
|AseI
|3 (Biolabs)
|3 (Biolabs)
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|✓
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|✗
|‘E - J61100-alkB2-J61100-rubA3 - P’
|‘E - J61100-alkB2-J61100-rubA3 - P’
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|-
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|HindIII
|HindIII
|2 (Biolabs)
|2 (Biolabs)
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|✓
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|✗
|‘E - J61100-rubA4-J61100-rubR-B0015 - P’
|‘E - J61100-rubA4-J61100-rubR-B0015 - P’
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|AseI
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|3 (Biolabs)
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|‘E - J61101-PhPFDα - P’
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|‘E - pSB1C3 - P’
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[[Image:|300px|thumb|left|Digestion check]]
[[Image:|300px|thumb|left|Digestion check]]
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==Ligation==
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Directly following the digestion, the products were [[Team:TU_Delft/protocols/ligation|ligated]] for 4 hours:
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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 / Plasmid'''
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|-
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|1
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|014K
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|‘E - J61100-alkB2-J61100-rubA3 - S’
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|‘E - J61100-rubA4-J61100-rubR-B0015-pSB1AK3 - X’
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|-
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|2
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|020A
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|‘E - J61100-ladA - S’
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|‘E - J61101-ADH-pSB1A2 - X’
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|-
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|3
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|011C
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|‘E - J61100-alkB2-J61100-rubA3 - P’
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|‘E - pSB1C3 - P’
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|-
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|4
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|013C
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|‘E - J61100-rubA4-J61100-rubR-B0015 - P’
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|‘E - pSB1C3 - P’
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|-
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|5
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|021C
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|‘E - J61107-ALDH-B0015 - P’
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|‘E - pSB1C3 - P’
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|-
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|5
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|402C
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|‘E - J61101-PhPFDα - P’
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|‘E - pSB1C3 - P’
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|-
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|5
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|403C
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|‘E - J61101-PhPFDβ - P’
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|‘E - pSB1C3 - P’
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|-
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|}
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10 μL of this ligation mix were used to [[Team:TU_Delft/protocols/transformation| transform]] Top10 competent cells.

Revision as of 12:21, 12 August 2010

Alkane degradation

BioBricks number 014K and 020A (already tries before but failed) are up today. We will do a digestion and a ligation followed by a transformation in order to make these BioBricks. Next to these two BioBricks we will also be working on placing some of the previously made BioBricks in the 1C3 plasmid so we can ship it to the registry.

Digestion

# Sample Enzyme 1 Enzyme 2 Enzyme 3 Buffer BSA Needed fragment
1 011A EcoRI SpeI AseI 2 (Biolabs) ✓ ‘E - J61100-alkB2-J61100-rubA3 - S’
2 013K EcoRI XbaI ✗ 3 (Biolabs) ✓ ‘E - J61100-rubA4-J61100-rubR-B0015-pSB1AK3 - X’
3 017A EcoRI SpeI AseI 2 (Biolabs) ✓ ‘E - J61100-ladA - S’
4 018A EcoRI XbaI ✗ 3 (Biolabs) ✓ ‘E - J61101-ADH-pSB1A2 - X’
5 011A EcoRI PstI AseI 3 (Biolabs) ✗ ‘E - J61100-alkB2-J61100-rubA3 - P’
6 013K EcoRI PstI HindIII 2 (Biolabs) ✗ ‘E - J61100-rubA4-J61100-rubR-B0015 - P’
7 021 EcoRI PstI SmaI 3 (Biolabs) ✓ ‘E - J61107-ALDH-B0015 - P’
8 402 EcoRI PstI AseI 3 (Biolabs) ✗ ‘E - J61101-PhPFDα - P’
9 403 EcoRI PstI AseI 3 (Biolabs) ✗ ‘’
10 pSB1C3 EcoRI PstI ✗ 3 (Biolabs) ✗ ‘E - pSB1C3 - P’

The digestions were done during 2 hours, and checked on a gel:

[[Image:|300px|thumb|left|Digestion check]]

Ligation

Directly following the digestion, the products were ligated for 4 hours:

# BioBrick Fragment 1 Fragment 2 / Plasmid
1 014K ‘E - J61100-alkB2-J61100-rubA3 - S’ ‘E - J61100-rubA4-J61100-rubR-B0015-pSB1AK3 - X’
2 020A ‘E - J61100-ladA - S’ ‘E - J61101-ADH-pSB1A2 - X’
3 011C ‘E - J61100-alkB2-J61100-rubA3 - P’ ‘E - pSB1C3 - P’
4 013C ‘E - J61100-rubA4-J61100-rubR-B0015 - P’ ‘E - pSB1C3 - P’
5 021C ‘E - J61107-ALDH-B0015 - P’ ‘E - pSB1C3 - P’
5 402C ‘E - J61101-PhPFDα - P’ ‘E - pSB1C3 - P’
5 403C ‘E - J61101-PhPFDβ - P’ ‘E - pSB1C3 - P’


10 μL of this ligation mix were used to transform Top10 competent cells.

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