Haynes Lab:Notebook/Engineering PC-TFs/2015/01/19: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
(Autocreate 2015/01/19 Entry for Haynes_Lab:Notebook/Engineering_PC-TFs)
 
(One intermediate revision by the same user not shown)
Line 7: Line 7:
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
==Summary==
==Summary==
*
'''Ligation 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><br>


| rowspan="10" | [[Image:1-20-2015 lcr lt remainder digest.JPG|400px|Today's gel]]<br>
15 μL/lane; 1% agarose;<br>
<u>Observed lengths:</u> <br>
W2 BL05 LCR SV1 (small volume lcr reaction product: 4uL) <br>
W3 BL05 LCR SV2<br>
W4 BL05 LCR LV1 (large volume lcr reaction product: 16uL)<br>
W5 BL05 LCR NEG<br>
W6 BL01 LT (Traditional Ligation) SV1 RED <br>
W7 BL01 LT SV2 RED <br>
W8 BL01 LT LV1 <br>
W9 BL01 LT LV2 <br>
W10 BL05 LT LV2<br>
W11 Empty CMV/MV9 <br>
W12 BL01 LT SV3 NOT RED<br>
W13 BL05 LT SV1<br>
W14 BL05 LT SV2<br>
[http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
| DNA[lanes 2-9*](lanes10-14)|| [8.0 μL] (4.0 μL)
|-
| 10X buffer || 1.5 μL
|-
| XbaI || 1.0 μL
|-
| BsmbI|| 0.5 μL
|-
| dH<sub>2</sub>O || [4μL] (8μL)
|-
| Total || 15 μL --> 37°C/ 30 min.
|}
<br>
*DNA in lanes 2-9 have relatively low concentration (<90ng/μL).<br>
Wells 6&7 (BL01 traditional ligation) look promising; will sequence tomorrow. <br>
First four lanes are empty yet again yet have reasonable concentration and purity in the plate reader. Degradation? <br>
Using DD123 & DD122 primers, a forward insertion will result in a band of length 3371 running the PCR reaction.


<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->

Revision as of 19:13, 19 January 2015



 		<html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

Summary

Ligation 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

 Today's gel

15 μL/lane; 1% agarose;
Observed lengths:
W2 BL05 LCR SV1 (small volume lcr reaction product: 4uL)
W3 BL05 LCR SV2
W4 BL05 LCR LV1 (large volume lcr reaction product: 16uL)
W5 BL05 LCR NEG
W6 BL01 LT (Traditional Ligation) SV1 RED
W7 BL01 LT SV2 RED
W8 BL01 LT LV1
W9 BL01 LT LV2
W10 BL05 LT LV2
W11 Empty CMV/MV9
W12 BL01 LT SV3 NOT RED
W13 BL05 LT SV1
W14 BL05 LT SV2
Ladder

DNA[lanes 2-9*](lanes10-14) [8.0 μL] (4.0 μL)
10X buffer 1.5 μL
XbaI 1.0 μL
BsmbI 0.5 μL
dH2O [4μL] (8μL)
Total 15 μL --> 37°C/ 30 min.


  • DNA in lanes 2-9 have relatively low concentration (<90ng/μL).

Wells 6&7 (BL01 traditional ligation) look promising; will sequence tomorrow.
First four lanes are empty yet again yet have reasonable concentration and purity in the plate reader. Degradation?
Using DD123 & DD122 primers, a forward insertion will result in a band of length 3371 running the PCR reaction.


Retrieved from "https://openwetware.org/mediawiki/index.php?title=Haynes_Lab:Notebook/Engineering_PC-TFs/2015/01/19&oldid=851624"