Team:University College London/Protocols/Week13/2

From 2012.igem.org

(Difference between revisions)
(Created page with "Ligations for irrE, nuclease, laccase & curli Digest upstream part with EcoRI-HF™ and SpeI {| class="bigtable" |- ! Constitutive promoter-rbs construct !! 500 ng |- | EcoRI-H...")
 
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| H<sub>2</sub>O || to 50 µl
| H<sub>2</sub>O || to 50 µl
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{| class="bigtable"
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|-
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! PSB1C3 !! 500 ng
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| EcoRI-HF || 1 µl
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| Pstl || 1 µl
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| 10X NEBuffer 2 || 5 µl
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|- 100X BSA || 0.5 µl
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|-
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| H<sub>2</sub>O || to 50 µl
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|}
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Incubate all three restriction digest reactions at 37°C for 10 minutes and then heat inactivate at 80°C for 20 minutes.
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Ligate the upstream and downstream parts into the digested destination plasmid.
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{| class="bigtable"
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|-
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| Upstream part digestion || 2 µl
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|-
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| Downstream part digestion || 2 µl
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|-
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| Destination plasmid digestion || 2 µl
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|-
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| 10X T4 DNA ligase buffer || 2 µl
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|-
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| T4 DNA ligase || 1 µl
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|-
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| H<sub>2</sub>O || 11 µl
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|}
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Incubate at room temperature for 10 minutes and then heat inactivate at 80°C for 20 minutes.
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Summary of the cuts:
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{| class="bigtable"
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|-
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! !! Part !! Enzymes
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| 1 || laccase || X+P
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|-
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| 2 || curli || X+P
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| 3 || irrE || X+P
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| 4 || irrE || E+P
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| 5 || nuclease || X+P
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| 6 || nuclease || X+P
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| 7 || plasmid backbone || E+P
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| 8 || plasmid backbone || E+P
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|-
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| 9 || linker || E+S
|}
|}

Latest revision as of 22:50, 26 September 2012

Ligations for irrE, nuclease, laccase & curli

Digest upstream part with EcoRI-HF™ and SpeI

Constitutive promoter-rbs construct 500 ng
EcoRI-HF 1µl
Spel 1 µl
10X NEBuffer 2 5 µl
100X BSA 0.5 µl
H2O to 50 µl

Digest downstream part with Xbal and Pstl

Curli (from PCR product)
Laccase (from PCR product)
irrE (from PCR product)
Nuclease (in puc57) 		500 ng
Xbal 1 µl
Pstl 1 µl
10X NEBuffer 2 5 µl
100X BSA 0.5 µl
H2O to 50 µl
PSB1C3 500 ng
EcoRI-HF 1 µl
Pstl 1 µl
10X NEBuffer 2 5 µl
H2O to 50 µl


Incubate all three restriction digest reactions at 37°C for 10 minutes and then heat inactivate at 80°C for 20 minutes.

Ligate the upstream and downstream parts into the digested destination plasmid.

Upstream part digestion 2 µl
Downstream part digestion 2 µl
Destination plasmid digestion 2 µl
10X T4 DNA ligase buffer 2 µl
T4 DNA ligase 1 µl
H2O 11 µl


Incubate at room temperature for 10 minutes and then heat inactivate at 80°C for 20 minutes.

Summary of the cuts:

Part Enzymes
1 laccase X+P
2 curli X+P
3 irrE X+P
4 irrE E+P
5 nuclease X+P
6 nuclease X+P
7 plasmid backbone E+P
8 plasmid backbone E+P
9 linker E+S
Retrieved from "http://2012.igem.org/Team:University_College_London/Protocols/Week13/2"