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

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==Summary==
==Summary==
* Restriction Digest/Gel Verification, Concentration Reading<br>
* Restriction Digest/Gel Verification, Concentration Reading<br>
'''Restriction Digest'''<br>


'''Digest and Gel Verification'''
<br>
{| {{table}} border="1" cellspacing="5"
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
| rowspan="9" |
<u><b>Expected:</b></u>
<br>
<u>BL01/5 Ligation Colonies [XbaI/NruI]</u><br>
Forward insertion: 3659, 4064 <br>
Reverse insertion: 1538, 6185 <br>
No insertion: 1538, 4064 <br> <br>
<u>CMV/MV9 with [XbaI/NruI] </u> <br>
1538, 4064<br>
<u> Uncut CMV/MV9:</u> 5197<br>
<u> Uncut CMV/BL05/MV9:</u> 7723 <br><br>


| rowspan="10" | [[Image:02 28 2015 bl05 gel.JPG|400px|Today's gel]]<br>
15 μL/lane; 1% agarose;<br>
<u>Observed lengths:</u> <br>
W1: Ladder<br>
W2: Ryan's stuff <br>
W3: BL05 LCR <br>
W4: BL05 LCR <br>
W5: BL05 LCR <br>
W6: BL05 LCR <br>
W7: BL05 LCR <br>
W8: BL05 LCR <br>
W9: BL05 LCR <br>
W10: BL05 LCR <br>
W11: BL05 LCR <br>
W12: BL05 LCR <br>
W13: BL05 LCR <br>
W14: BL05 LCR <br>
[http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
| DNA|| 8.0 μL
|-
| 10X buffer || 1.5 μL
|-
| XbaI || 1.0 μL
|-
| BsmbI|| 0.5 μL
|-
| dH<sub>2</sub>O || 4 μL
|-
| Total || 15 μL --> 37°C/ 20 min.
|}
<br>
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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Revision as of 09:58, 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 8.0 μL
10X buffer 1.5 μL
XbaI 1.0 μL
BsmbI 0.5 μL
dH2O 4 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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