Haynes Lab:Notebook/Engineering PC-TFs/2015/01/28: Difference between revisions

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(Autocreate 2015/01/28 Entry for Haynes_Lab:Notebook/Engineering_PC-TFs)
 
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==Summary==
==Summary==
*
==Summary==
'''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:1-27-2015 streak plate digest bl05.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 Colony 11<br>
W4: '' Colony 13<br>
W5: '' Colony 17<br>
W6: '' Colony 19<br>
W7: BL05 Traditional Ligation Colony 32<br>
W8: '' Colony 33<br>
W9: '' Colony 36<br>
W10: '' Colony 37<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>
Rather inconclusive without empty CMV/MV9. Again I'm not sure why several lanes appear empty when they have high concentration and purity given plate reader data. Will re-run with empty CMV/MV9 as wells 5&6 could have forward insertion (or uncut CMV/MV9 & no insertion bands).




Revision as of 09:24, 28 January 2015



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Summary

Summary

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 Colony 11
W4: Colony 13
 W5: Colony 17
 W6: Colony 19
 W7: BL05 Traditional Ligation Colony 32
W8: Colony 33
 W9: Colony 36
 W10: Colony 37
 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.


Rather inconclusive without empty CMV/MV9. Again I'm not sure why several lanes appear empty when they have high concentration and purity given plate reader data. Will re-run with empty CMV/MV9 as wells 5&6 could have forward insertion (or uncut CMV/MV9 & no insertion bands).



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