Team:KIT-Kyoto/kazukokekokko

From 2012.igem.org

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<h2>BP reaction by invitrogen gateway system</h2>
<h2>BP reaction by invitrogen gateway system</h2>
<br>
<br>
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1. PCR using promers containing the attB sequence.
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1. PCR using primers containing the attB sequence.
<br>
<br>
-
*attB sequence:
+
2.Purify PCR product.
-
<br>
+
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2. Purification PCR products.
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<br>
<br>
3. Prepare vials in 1.5mL tube according to the following components.
3. Prepare vials in 1.5mL tube according to the following components.
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</Table>
</Table>
<br>
<br>
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4. Dissolve BP Clonase Ⅱ enzyme mix on ice.
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4. Melt BP Clonase Ⅱ enzyme mix on ice.
<br>
<br>
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5. Add BP Clonase Ⅱ enzyme mix (2uL or 1uL) to vials, vortex and spin them down.
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5. Add BP Clonase Ⅱ enzyme mix (2uL or 1uL) to tube, vortex and spin them down.
<br>
<br>
6. Incubate vials at 25℃ for more than an hour.
6. Incubate vials at 25℃ for more than an hour.
<br>
<br>
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7. Add Proteinase K solutionto vials (1uL) and vortex shortly.
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7. Add Proteinase K solutionton (1uL) vials and vortex briefly.
<br>
<br>
8. Incubate at 37℃ for 10 minutes.
8. Incubate at 37℃ for 10 minutes.
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</Table>
</Table>
<br>
<br>
-
2. Dissolve LR Clonase Ⅱ enzyme mix on ice  
+
2.Melt LR Clonase Ⅱ enzyme mix on ice  
<br>
<br>
-
3. Add LR Clonase Ⅱ enzyme mix to vials by 2uL or 1uL, votex and spin down.  
+
3. Add LR Clonase Ⅱ enzyme mix to tube (2uL or 1uL), votex and spin down.  
<br>
<br>
4. Incubate vials at 25℃ for more than an hour.
4. Incubate vials at 25℃ for more than an hour.
<br>
<br>
-
5. Add Proteinase K solution to vials by 1uL and vortex it shortly.
+
5. Add Proteinase K solution (1μl) to vials and vortex it briefly.
<br>
<br>
6. Incubate at 37℃ for 30 minutes.
6. Incubate at 37℃ for 30 minutes.

Revision as of 08:39, 16 September 2012







BP reaction by invitrogen gateway system


1. PCR using primers containing the attB sequence.
2.Purify PCR product.
3. Prepare vials in 1.5mL tube according to the following components.

attB DNA sample75ng/reaction
pDONR vector150ng/reaction
TE Bufferup
Total8uL or 9uL

4. Melt BP Clonase Ⅱ enzyme mix on ice.
5. Add BP Clonase Ⅱ enzyme mix (2uL or 1uL) to tube, vortex and spin them down.
6. Incubate vials at 25℃ for more than an hour.
7. Add Proteinase K solutionton (1uL) vials and vortex briefly.
8. Incubate at 37℃ for 10 minutes.

LR reaction by invitrogen gateway system


1. Control vials in 1.5mL tube according to the following components.

BP DNA sample50-150ng/reaction
Destination vector150ng/reaction
TE Bufferup
Total8uL or 9uL

2.Melt LR Clonase Ⅱ enzyme mix on ice
3. Add LR Clonase Ⅱ enzyme mix to tube (2uL or 1uL), votex and spin down.
4. Incubate vials at 25℃ for more than an hour.
5. Add Proteinase K solution (1μl) to vials and vortex it briefly.
6. Incubate at 37℃ for 30 minutes.


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