Haynes Lab:Notebook/Engineering PC-TFs/2014/08/01

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(Summary)
Current revision (15:47, 8 August 2014) (view source)
(Summary)
 
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[http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
[http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
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| DNA(BL01x2, BL05x1, BL09x2, CMV/MV9x1) || 3.0 μL
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| DNA(BL09x3, CMV/MV9x3) || 3.0 μL
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| 10X buffer || 1.5 μL
| 10X buffer || 1.5 μL
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| XbaI || 1.0 μL
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| XbaI || 1.0 μL(or 0 for dephos CMV/MV9)
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| NruI || 1.0 μL
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| NruI || 1.0 μL (or 0 CMV/MV9 and dephos CMV/MV9)
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| dH<sub>2</sub>O || 8.5 μL
| dH<sub>2</sub>O || 8.5 μL

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Summary

Ligation Digest and Gel Verification

Reagent Volume

Expected:
BL09 Ligation Colonies 1-3[XbaI/NruI] (Well: 2-4)
Forward insertion: 710, 5921
Reverse insertion: 2141, 4490
No insertion: 710, 4490

CMV/MV9 with [XbaI/NruI] (Well: 5)
710, 4490

CMV/MV9 [XbaI](Well: 6)
5200

Dephos CMV/MV9 (Well: 7)
5200

Today's gel

15 μL/lane; 1% agarose;
Observed lengths:
W2(BL09Lig1): 5000, 7100, 2500;
W3(BL09Lig2): 5000, 7100, 2500;
W4(BL09Lig3): 5000, 7100, 2500;
W5(Empty CM9): 5000, 700;
W6(CM9XbaI): 10000, 20000, 5000.
W7(DephosCM9): 5000
Ladder

DNA(BL09x3, CMV/MV9x3) 3.0 μL
10X buffer 1.5 μL
XbaI 1.0 μL(or 0 for dephos CMV/MV9)
NruI 1.0 μL (or 0 CMV/MV9 and dephos CMV/MV9)
dH2O 8.5 μL
Total 15 μL --> 37°C/ 30 min.




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