Haynes Lab:Notebook/Engineering PC-TFs/2014/07/28: Difference between revisions

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* Passaged cells.
* Passaged cells.
* Digested/dephos'd CMV/MV9.
* 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 -->
<!-- &nbsp; 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; [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 elution buffer 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. ##### -->

Revision as of 12:39, 29 July 2014



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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)

Reagent Volume Expected:
BL01(Well:2&3), CMV/MV9 (Well: 4) BL05(Well:5), BL09 (Well: 6&7)
Forward insertion: 710, 7010.
Reverse insertion: 3230, 4490.
No insertion: 710, 4490.
BL09 (W2:4-6)
Forward insertion: 710, 5921
Reverse insertion: 2141, 4490
No insertion: 710, 4490.
 Today's gel
15 μL/lane; 1% agarose;
Observed lengths: W2: 710, 4490; W3: 710, 4490; W4: 710, 4490; W5: 710, 4490; W6: 710, 4490.
W7: 7200, 5000, 2500
Ladder
DNA(BL01x2, BL05x1, BL09x2, CMV/MV9x1) 3.0 μL
10X buffer 1.5 μL
XbaI 1.0 μL
NruI 1.0 μL
dH2O 8.5 μL
Total 15 μL --> 37°C/ 30 min.


Sequencing Results Order 9218 (update when receive new sequencing data)


BL01rA

  • Used 1μL BL01 miniprep 1, 1μL DD123 B primer, 8μLdH2O.
  • Results: Matches were found after the primer and then just before CMV suggesting no insert.


BL01rB

  • Used 1μL BL01 miniprep 2, 1μL DD123 B primer, 8μLdH2O.
  • Results: Matches were found after the primer and then just before CMV suggesting no insert.


BL09rA

  • Used 1μL BL09 miniprep 1, 1μL DD123 B primer, 8μLdH2O.
  • Results: Matches were found after the primer and then just before CMV suggesting no insert.


Gel Extraction/Purification


Reagent Volume Expected:
1. BL01, BL05 = 2500bp, BL09=1400bp
DNA 25.0 μL
10X buffer 3.0 μL
XbaI 1.0 μL
SpeI 1.0 μL
dH2O 0 μL
Total 30 μL --> 37°C/ 35 min.


Hover name
0.8% agarose; cut the brightest band furthest down at ~ 2500bp; 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 elution buffer to the column.
  • Spun at max rpm for 30 seconds.


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