Haynes Lab:Notebook/Engineering PC-TFs/2015/02/26: Difference between revisions

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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|| 8.0 μL
| DNA|| 4.0 μL
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| 10X buffer || 1.5 μL
| 10X buffer || 1.5 μL
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| BsmbI|| 0.5 μL  
| BsmbI|| 0.5 μL  
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| dH<sub>2</sub>O || 4 μL  
| dH<sub>2</sub>O || 8 μL  
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| Total || 15 μL --> 37°C/ 20 min.
| Total || 15 μL --> 37°C/ 20 min.

Revision as of 10:01, 26 March 2015



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Summary

  • Restriction Digest/Gel Verification, Concentration Reading

Digest and Gel Verification

Reagent Volume

Expected:
BL01/5 Ligation Colonies [XbaI/NruI]
Forward insertion: 3659, 4064
Reverse insertion: 1538, 6185
No insertion: 1538, 4064

CMV/MV9 with [XbaI/NruI]
1538, 4064
Uncut CMV/MV9: 5197
Uncut CMV/BL05/MV9: 7723

Today's gel

15 μL/lane; 1% agarose;
Observed lengths:
W1: Ladder
W2: Ryan's stuff
W3: BL05 LCR
W4: BL05 LCR
W5: BL05 LCR
W6: BL05 LCR
W7: BL05 LCR
W8: BL05 LCR
W9: BL05 LCR
W10: BL05 LCR
W11: BL05 LCR
W12: BL05 LCR
W13: BL05 LCR
W14: BL05 LCR
Ladder

DNA 4.0 μL
10X buffer 1.5 μL
XbaI 1.0 μL
BsmbI 0.5 μL
dH2O 8 μL
Total 15 μL --> 37°C/ 20 min.


Looks as though one enzyme perhaps didn't cut or was not added. Empty wells probably a result of bad amp. DNA in well 13 will be re-examined as it seems to be bigger than the others. Possible insertion.


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