Haynes Lab:Notebook/Engineering PC-TFs/2015/04/20: Difference between revisions

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==Summary==
==Summary==
*  
* Restriction Digest/Gel Verification, Concentration Reading<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 Ligation Colonies [XbaI/ApaLI]</u><br>
Forward insertion: 255, 288, 883, 1243, 2037, 3017 <br>
Reverse insertion: 288, 255, 1122, 1243, 2037, 2783 <br>
No insertion: 268, 883, 1243, 2783 <br> <br>
<u>BL09 Ligation Colonies [XbaI/ApaLI]</u><br>
Forward insertion: 255, 288, 883, 1243, 3977 <br>
Reverse insertion: 255, 288, 1243, 2082, 2783 <br>
No insertion: 268, 883, 1243, 2783 <br> <br>
| rowspan="10" | [[Image:4-20-15.jpg|400px|]]<br>
15 μL/lane; 1% agarose;<br>
<u>Observed lengths:</u> <br>
W1: Ladder <br>
W2: Bl09 Lig 2 <br>
W3: Bl09 TL V1 <br>
W4: BL09 Lig 5 <br>
W5: "Bl01 LCR V3" <br>
W6: "BL01 LSR V4" <br>
W7: 1 LCR <br>
W8: Bl01 LCR V1 <br>
[http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
| DNA|| 2.5 μL  2.5 μL  2.0 μL  1.0 μL  1.0 μL  3.0 μL  2.0 μL (From ladder 2 to ladder 8)
|-
| 10X buffer || 2.0 μL (for all)
|-
| XbaI || 1.0 μL (for all)
|-
| ApaLI|| 1.0 μL  (for all)
|-
| dH<sub>2</sub>O || 8.5 μL  8.5 μL  9.5 μL  9.5 μL  10.5 μL  10.5 μL
|-
| Total || 15 μL --> 37°C/ 15 min.
|}
<br>
Note: Too much buffer was used. Messed up Well 7. As expected, "Bl01 LCR V3" and "BL01 LSR V4" are actually re-transformations of Bl09, they were just labeled incorrectly.
----
'''New PcTF Assembly'''<br>
PCD, AD, RFP in V0120.<br>
Either PCR or cut and paste biobrick assembly.<br>
Biobrick assembly:<br>
PCD: Cut out with Eco&Not<br>
RFP: Cut out with Not&Spe<br>
AD: Cut out with XbaI&PstI<br>
Ligate and then cut with XbaI and SpeI to make compatible with MV10 insertion site.


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Latest revision as of 00:55, 27 September 2017



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Summary

  • Restriction Digest/Gel Verification, Concentration Reading

Digest and Gel Verification

Reagent Volume

Expected:
BL01 Ligation Colonies [XbaI/ApaLI]
Forward insertion: 255, 288, 883, 1243, 2037, 3017
Reverse insertion: 288, 255, 1122, 1243, 2037, 2783
No insertion: 268, 883, 1243, 2783

BL09 Ligation Colonies [XbaI/ApaLI]
Forward insertion: 255, 288, 883, 1243, 3977
Reverse insertion: 255, 288, 1243, 2082, 2783
No insertion: 268, 883, 1243, 2783


15 μL/lane; 1% agarose;
Observed lengths:
W1: Ladder
W2: Bl09 Lig 2
W3: Bl09 TL V1
W4: BL09 Lig 5
W5: "Bl01 LCR V3"
W6: "BL01 LSR V4"
W7: 1 LCR
W8: Bl01 LCR V1

Ladder

DNA 2.5 μL 2.5 μL 2.0 μL 1.0 μL 1.0 μL 3.0 μL 2.0 μL (From ladder 2 to ladder 8)
10X buffer 2.0 μL (for all)
XbaI 1.0 μL (for all)
ApaLI 1.0 μL (for all)
dH2O 8.5 μL 8.5 μL 9.5 μL 9.5 μL 10.5 μL 10.5 μL
Total 15 μL --> 37°C/ 15 min.


Note: Too much buffer was used. Messed up Well 7. As expected, "Bl01 LCR V3" and "BL01 LSR V4" are actually re-transformations of Bl09, they were just labeled incorrectly.

New PcTF Assembly
PCD, AD, RFP in V0120.
Either PCR or cut and paste biobrick assembly.
Biobrick assembly:
PCD: Cut out with Eco&Not
RFP: Cut out with Not&Spe
AD: Cut out with XbaI&PstI
Ligate and then cut with XbaI and SpeI to make compatible with MV10 insertion site.


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