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

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==Summary==
==Summary==
'''Ligation Digest and Gel Verification (2)'''
'''Ligation Digest and Gel Verification'''
<br>
<br>
{| {{table}} border="1" cellspacing="5"  
{| {{table}} border="1" cellspacing="5"  
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| bgcolor=#cfcfcf | Volume
| bgcolor=#cfcfcf | Volume
| rowspan="9" |  
| rowspan="9" |  
<u>Expected:</u>
<u><b>Expected:</b></u>
<br>
<br>
BL09 Ligation Colonies 1-3[XbaI/NruI] (Well: 2-4)<br>  
<u>BL09 Ligation Colonies 1-3[XbaI/NruI] (Well: 2-4)</u><br>  
Forward insertion: 710, 5921 <br>
Forward insertion: 710, 5921 <br>
Reverse insertion: 2141, 4490 <br>
Reverse insertion: 2141, 4490 <br>
No insertion: 710, 4490<br> <br>
No insertion: 710, 4490<br> <br>
CMV/MV9 with [XbaI/NruI] (Well: 5) <br>
<u>CMV/MV9 with [XbaI/NruI] (Well: 5)</u> <br>
710, 4490<br><br>
710, 4490<br><br>
CMV/MV9 [XbaI](Well: 6) <br>
<u>CMV/MV9 [XbaI](Well: 6)</u> <br>
5200<br><br>
5200<br><br>
Dephos CMV/MV9 (Well: 7) <br>
<u>Dephos CMV/MV9 (Well: 7)</u> <br>
5200
5200
| rowspan="10" | [[Image:20140801 132828.jpg|400px|Today's gel]]<br>
| rowspan="10" | [[Image:20140801 132828.jpg|400px|Today's gel]]<br>
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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]
|-
|-
| DNA(BL01x2, BL05x1, BL09x2, CMV/MV9x1) || 3.0 μL
| DNA(BL09x3, CMV/MV9x3) || 3.0 μL
|-
|-
| 10X buffer || 1.5 μL
| 10X buffer || 1.5 μL
|-
|-
| XbaI || 1.0 μL
| XbaI || 1.0 μL(or 0 for dephos CMV/MV9)
|-
|-
| NruI || 1.0 μL
| NruI || 1.0 μL (or 0 CMV/MV9 and dephos CMV/MV9)
|-
|-
| dH<sub>2</sub>O || 8.5 μL
| dH<sub>2</sub>O || 8.5 μL

Revision as of 13:47, 8 August 2014



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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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