Haynes Lab:Notebook/Engineering PC-TFs/2014/09/01: Difference between revisions

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|style="background-color: #FFCC00"|<br>[[Image:Hayneslab3.gif|200px|center]][[Image:Asu logo 3.gif|200px|center]]<span style="font-size:22px;"><br></span>
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* Restriction digest/gel verification of PcTF CMV/MV9 ligation.
* Restriction digest/gel verification of PcTF CMV/MV9 ligation.
----
----
''''Restriction Digest/Gel Verification''''
'''Restriction Digest/Gel Verification'''
{| {{table}} border="1" cellspacing="5"  
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<u><b>Expected:</b></u>
<u><b>Expected:</b></u>
<br>
<br>
<br>BL01(Well: 4-8, 10-13), BL05(Well:14) <br>
<br>BL01(Well: 4-8, 11-14), BL05(Well:2) <br>
Forward insertion: 710, 7010. <br>
Forward insertion: 710, 7010. <br>
Reverse insertion: 3230, 4490. <br>
Reverse insertion: 3230, 4490. <br>
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| rowspan="10" | [[IMAGE URL HERE|400px|Today's gel]]<br>
| rowspan="10" | [[Image:20140901 180224.jpg|400px|Today's gel]]<br>
15 μL/lane; 1% agarose;<br>  
15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]<br>  


<u>Observed lengths:</u> <br>
<u>Observed lengths:</u> <br>
W2(BL01Lig1): 5000, 700; <br>
W2(BL05Lig1): 5000, 700; <br>
W3(BL05Lig1): 5000, 700; <br>
W4(BL01Lig1): 3200, 4500; <br>
W4(BL05Lig2): 5000, 700; <br>
W5(BL01Lig2): 5000, 700; <br>
W5(BL05Lig3): 5000, 700; <br>
W6(BL01Lig3): 5000, 700; <br>  
W6(BL05Lig4): 5000, 700; <br>  
W7(BL01Lig4): 5000, 700; <br>  
W7(Empty CMV/MV9): 5000, 700; <br>  
W8(BL01Lig5): 5000, 700; <br>  
W8(BL09Lig1): 5000, 700; <br>  
W9(Empty CMV/MV9): 5000, 700; <br>  
W9(BL09Lig2): 7000, 700; <br>  
W11(BL01Lig6): 5000, 700; <br>  
W10(BL09Lig3): 5000, 700; <br>  
W12(BL01Lig7): 5000, 700; <br>  
W11(BL09Lig4): 5000, 700; <br>  
W13(BL01Lig8): 5000, 700;<br>  
W12(BL09Lig5): 7000, 700;<br>  
W14(BL01Lig9): 5000, 700; <br>  
W13(BL09Lig6): 5000, 700; <br>  
[http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
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| DNA(BL01x2, BL05x1, BL09x2, CMV/MV9x1) || 2.0 μL
| DNA(BL01x2, BL05x1, BL09x2, CMV/MV9x1) || 2.0 μL
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| Total || 15 μL --> 37°C/ 30 min.
| Total || 15 μL --> 37°C/ 30 min.
|}
|}
 
<br>
One reverse insertion. No forward insertions. Will reattempt ligation.


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Latest revision as of 00:13, 27 September 2017



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Summary

  • Split cells.
  • Restriction digest/gel verification of PcTF CMV/MV9 ligation.

Restriction Digest/Gel Verification

Reagent Volume

Expected:

BL01(Well: 4-8, 11-14), BL05(Well:2)
Forward insertion: 710, 7010.
Reverse insertion: 3230, 4490.
No insertion: 710, 4490.


CMV/MV9 [XbaI/NruI] (Well: 9)
710, 4490


 Today's gel

15 μL/lane; 1% agarose; Ladder

Observed lengths:
W2(BL05Lig1): 5000, 700;
W4(BL01Lig1): 3200, 4500;
W5(BL01Lig2): 5000, 700;
W6(BL01Lig3): 5000, 700;
W7(BL01Lig4): 5000, 700;
W8(BL01Lig5): 5000, 700;
W9(Empty CMV/MV9): 5000, 700;
W11(BL01Lig6): 5000, 700;
W12(BL01Lig7): 5000, 700;
W13(BL01Lig8): 5000, 700;
W14(BL01Lig9): 5000, 700;

DNA(BL01x2, BL05x1, BL09x2, CMV/MV9x1) 2.0 μL
10X buffer 1.5 μL
XbaI 1.0 μL
NruI 1.0 μL
dH2O 9.5 μL
Total 15 μL --> 37°C/ 30 min.


One reverse insertion. No forward insertions. Will reattempt ligation.


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