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
Digest and Gel Verification
New PcTF Assembly |