Haynes Lab:Notebook/Engineering PC-TFs/2014/07/28: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
(Autocreate 2014/07/28 Entry for Haynes_Lab:Notebook/Engineering_PC-TFs) |
(fix raw html notebook nav) |
||
(3 intermediate revisions by one other user not shown) | |||
Line 2: | Line 2: | ||
|- | |- | ||
|style="background-color: #FFCC00"|<br>[[Image:Hayneslab3.gif|200px|center]][[Image:Asu logo 3.gif|200px|center]]<span style="font-size:22px;"><br></span> | |style="background-color: #FFCC00"|<br>[[Image:Hayneslab3.gif|200px|center]][[Image:Asu logo 3.gif|200px|center]]<span style="font-size:22px;"><br></span> | ||
|style="background-color: #800000" align="center"| | |style="background-color: #800000" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
|- | |- | ||
| colspan="2"| | | colspan="2"| | ||
<!-- ##### 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== | ||
* | * Ran another gel verification on prior ligation results. | ||
* Gel extraction/purification for BL01, BL05, BL09. | |||
* Passaged cells. | |||
* Digested/dephos'd CMV/MV9. | |||
* Resubmitted samples for sequencing to biodesign. | |||
---- | |||
'''Ligation Digest and Gel Verification (2)''' | |||
<br> | |||
{| {{table}} border="1" cellspacing="5" | |||
|- valign="top" | |||
| bgcolor=#cfcfcf | Reagent | |||
| bgcolor=#cfcfcf | Volume | |||
| rowspan="9" | <u>Expected:</u><br>BL01(Well:2&3), CMV/MV9 (Well: 4) BL05(Well:5), BL09 (Well: 6&7) <br>Forward insertion: 710, 7010. <br>Reverse insertion: 3230, 4490. <br>No insertion: 710, 4490.<br> BL09 (W2:4-6) <br>Forward insertion: 710, 5921 <br>Reverse insertion: 2141, 4490 <br>No insertion: 710, 4490.<br> | |||
| rowspan="10" | [[Image:Ligation gel ver2 20140728 171828.jpg|400px|Today's gel]]<br>15 μL/lane; 1% agarose;<br> <u>Observed lengths:</u> W2: 710, 4490; W3: 710, 4490; W4: 710, 4490; W5: 710, 4490; W6: 710, 4490.<br> W7: 7200, 5000, 2500<br> [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | |||
|- | |||
| DNA(BL01x2, BL05x1, BL09x2, CMV/MV9x1) || 3.0 μL | |||
|- | |||
| 10X buffer || 1.5 μL | |||
|- | |||
| XbaI || 1.0 μL | |||
|- | |||
| NruI || 1.0 μL | |||
|- | |||
| dH<sub>2</sub>O || 8.5 μL | |||
|- | |||
| Total || 15 μL --> 37°C/ 30 min. | |||
|} | |||
<br> | |||
'''Sequencing Results Order 9218 (update when receive new sequencing data)''' | |||
---- | |||
<br> | |||
<u>BL01rA<br></u> | |||
*Used 1μL BL01 miniprep 1, 1μL DD123 B primer, 8μLdH<sub>2</sub>O. | |||
*'''Results''': Matches were found after the primer and then just before CMV suggesting no insert. | |||
<br> | |||
<u>BL01rB</u><br> | |||
*Used 1μL BL01 miniprep 2, 1μL DD123 B primer, 8μLdH<sub>2</sub>O. | |||
*'''Results''': Matches were found after the primer and then just before CMV suggesting no insert. | |||
<br> | |||
<u>BL09rA</u><br> | |||
*Used 1μL BL09 miniprep 1, 1μL DD123 B primer, 8μLdH<sub>2</sub>O. | |||
*'''Results''': Matches were found after the primer and then just before CMV suggesting no insert. | |||
<br> | |||
'''Gel Extraction/Purification''' | |||
---- | |||
<br> | |||
{| {{table}} border="1" cellspacing="3" | |||
<!-- Editing: the coding for this table is a bit more advanced. --> | |||
<!-- valign="top" aligns all the text in the first row to the top. --> | |||
<!-- The | symbols on the next two lines start new cells in the same row. This does the same thing as ||, but you have to use | on new lines to set formatting for cells. --> | |||
<!-- bgcolor=#cfcfcf *colors* the *background* of the Reagent and Volume cells grey. --> | |||
<!-- The next two "rowspan=7" cells span all 7 rows in the table so that they can fit the "Expected" list and a gel image. rowspan is how you create merged rows in Wiki code. --> | |||
<!-- Replace Plasmid 1 and 2 with the names of your plasmids. Replace insert size and vector size with appropriate information for your plasmids. If you only have one Plasmid, delete all the text for Plasmid 2 | |||
<!-- After you upload your gel image to OWW, replace "GelImage.jpg" with the name of your image file --> | |||
<!-- is ASCII code for an invisible space. --> | |||
|- valign="top" | |||
| bgcolor=#cfcfcf | Reagent | |||
| bgcolor=#cfcfcf | Volume | |||
| rowspan="7" | <u>Expected:</u><br>1. BL01, BL05 = 2500bp, BL09=1400bp <br> | |||
|- | |||
| DNA || 25.0 μL | |||
|- | |||
| 10X buffer || 3.0 μL | |||
|- | |||
| XbaI || 1.0 μL | |||
|- | |||
| SpeI || 1.0 μL | |||
|- | |||
| dH<sub>2</sub>O || 0 μL | |||
|- | |||
| Total || 30 μL --> 37°C/ 35 min. | |||
|} | |||
<br> | |||
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | |||
|- valign="top" | |||
| rowspan="7" | [[Image:Pctf gel extraction 20140728 180402.jpg|400px|Hover name]]<br> 0.8% agarose; cut the brightest band furthest down at ~ 2500bp&1400bp; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | |||
|} | |||
* Used Zymo gel purification kit. | |||
* Assumed 200mg gel, added 600μL ADB to the 1.5mL tube. | |||
* Put on heat block at 55°C for 5 minutes, vortexed and then placed back on the heat block for an additional 5 minutes, vortexed again. | |||
* Put ~750μL gel/ADB into spin column, centrifuged at max rpm for 30 seconds. | |||
* Pipetted 200μL wash buffer to the tube, centrifuged at max rpm for 30 seconds. Repeated once. | |||
* Transferred column to new labeled 1.5mL tube. | |||
* Pipetted 15μL dH<sub>2</sub>O to the column. | |||
* Spun at max rpm for 30 seconds. | |||
<!-- ##### 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. ##### --> |
Latest revision as of 00:08, 27 September 2017
Main project page Previous entry Next entry | |||||||||||||||||||||||||||||||||
Summary
Ligation Digest and Gel Verification (2)
Gel Extraction/Purification
|