Team:TU Delft/29 July 2010 content
From 2010.igem.org
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==Alkane Degradation== | ==Alkane Degradation== | ||
- | + | ====PCR Amplification==== | |
K398007A, K398008A, K398009A, K398010A, K398017A, K398018A, K398019A, B0015 were [[Team:TU_Delft/protocols/PCR|amplified]] with the universal primers G00100 and G00101. The product was put on [[Team:TU_Delft/protocols/agarose_gel|1% agarose gel]]: | K398007A, K398008A, K398009A, K398010A, K398017A, K398018A, K398019A, B0015 were [[Team:TU_Delft/protocols/PCR|amplified]] with the universal primers G00100 and G00101. The product was put on [[Team:TU_Delft/protocols/agarose_gel|1% agarose gel]]: | ||
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|1 | |1 | ||
- | | | + | |K398011 |
|4 μL ‘E–007–S’ | |4 μL ‘E–007–S’ | ||
- | |4 μL | + | |4 μL ‘X–008–P’ |
- | | | + | |10 μL |
|- | |- | ||
|2 | |2 | ||
- | | | + | |K398012 |
- | | | + | |4 μL ‘E–009–S’ |
- | | | + | |4 μL ‘X–010–P’ |
- | | | + | |10 μL |
|- | |- | ||
|3 | |3 | ||
- | | | + | |K398020 |
- | | | + | |4 μL ‘E–017–S’ |
- | | | + | |4 μL ‘X–018–P’ |
- | | | + | |10 μL |
|- | |- | ||
|4 | |4 | ||
- | | | + | |K398021 |
- | | | + | |4 μL ‘E–019–S’ |
- | | | + | |4 μL ‘X–B0015–P’ |
- | | | + | |10 μL |
|- | |- | ||
|5 | |5 | ||
- | | | + | |negative control |
- | | | + | |4 μL ‘E–007–S’ |
- | | | + | |4 μL H2O |
- | | | + | |10 μL |
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|} | |} | ||
- | + | ====Plasmid isolation==== | |
- | A plasmid isolation was done on the positive colonies of [https://2010.igem.org/Team:TU_Delft#/blog?blog=28_July_2010 yesterday]. This was done using a | + | A plasmid isolation was done on the positive colonies of [https://2010.igem.org/Team:TU_Delft#/blog?blog=28_July_2010 yesterday]. This was done using a [[Team:TU_Delft/protocols/mini-prep_plasmid_isolation|Qiagen Miniprep kit]]. We used 2 mL of the bacterial cells to make [[Team:TU_Delft/protocols/freezing_bacterial_stocks|-80 °C stocks]]. |
Revision as of 19:42, 2 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 | Remarks |
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 | ✓ |
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’ |
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.