Haynes Lab:Notebook/Engineering PC-TFs/2014/03/22: Difference between revisions

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(Autocreate 2014/03/22 Entry for Haynes_Lab:Notebook/Engineering_PC-TFs)
 
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==Summary==
==Summary==
*  
* Miniprep on ligated plasmid CMV MV9 liquid culture.
* Gel purification from prior CMV miniprep.
----
''' Performed mini-prep on each of the four picked colonies.''' <br>
* Ran concentration check on each:
 
{| {{table}}
|- bgcolor=#cfcfcf
| Plasmid || OD260 || OD260/280 || ng/μL
|-
| 1. Colony 1 CMV || 0.005 || 1.211 || 4.895
|-
| 2. Colony 2 CMV || 0.003 || 1.273 || 2.963
|-
| 3. Colony 3 CMV || 0.034 || 1.888 || 33.572
|-
| 4. Colony 4 CMV || 0.007 || -5.308 || 7.215
|}
 
 
----
 
'''Digest CMV''' <br>
 


'''Restriction Digest Table'''<br>
* Checked plasmid minipreps with EcoRI/PstI digests
{| {{table}} border="1" cellspacing="3"
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<!-- 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 -->
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|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
| rowspan="7" | <u>Expected:</u><br>1. CMV = 588bp <br>
|-
| DNA(CMV) || 25.0 μL
|-
| 10X buffer || 3.0 μL
|-
| EcoRI || 1.0 μL
|-
| PstI || 1.0 μL
|-
| dH<sub>2</sub>O || 0 μL
|-
| &nbsp; || 15 μL --> 37°C/ 15 min.
|}
----
''' Gel Purification: CMV/V0120 ''' <br>
* Using Prior Miniprep Digests <br>
# CMV culture 1 - check with E/P
* Used previously digested CMV.
* Placed 15μL 1kB Ladder in well one and 30μL of CMV digest in well two.
* Ran gel at 110V for 30 minutes.
* Placed gel on UV transilluminator and cut out desired bands.
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
|- valign="top"
| rowspan="7" | [[Image:2014-03-22 10.07.35.jpg|400px|Hover name]]<br>15 μL/lane; 1% agarose; cut the brightest band furthest down at ~ 588bp; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|}
* Used Zymo gel purification kit.
* Assumed 200mg gel, added 600μL ADB to each 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 each tube, centrifuged at max rpm for 30 seconds. Repeated once.
* Transferred columns to new labeled 1.5mL tube.
* Pipetted 15μL elution buffer to each column.
* Spun at max rpm for 30 seconds.
* Put marked (CMV gel pur2 3/22) CMV in -20°C fridge in box labeled Cameron.
----


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__NOTOC__
__NOTOC__

Revision as of 14:35, 22 March 2014



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Summary

  • Miniprep on ligated plasmid CMV MV9 liquid culture.
  • Gel purification from prior CMV miniprep.

Performed mini-prep on each of the four picked colonies.

  • Ran concentration check on each:
Plasmid OD260 OD260/280 ng/μL
1. Colony 1 CMV 0.005 1.211 4.895
2. Colony 2 CMV 0.003 1.273 2.963
3. Colony 3 CMV 0.034 1.888 33.572
4. Colony 4 CMV 0.007 -5.308 7.215


Digest CMV


Restriction Digest Table

  • Checked plasmid minipreps with EcoRI/PstI digests
Reagent Volume Expected:
1. CMV = 588bp
DNA(CMV) 25.0 μL
10X buffer 3.0 μL
EcoRI 1.0 μL
PstI 1.0 μL
dH2O 0 μL
  15 μL --> 37°C/ 15 min.


Gel Purification: CMV/V0120

  • Using Prior Miniprep Digests
  1. CMV culture 1 - check with E/P
  • Used previously digested CMV.
  • Placed 15μL 1kB Ladder in well one and 30μL of CMV digest in well two.
  • Ran gel at 110V for 30 minutes.
  • Placed gel on UV transilluminator and cut out desired bands.
Hover name
15 μL/lane; 1% agarose; cut the brightest band furthest down at ~ 588bp; Ladder
  • Used Zymo gel purification kit.
  • Assumed 200mg gel, added 600μL ADB to each 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 each tube, centrifuged at max rpm for 30 seconds. Repeated once.
  • Transferred columns to new labeled 1.5mL tube.
  • Pipetted 15μL elution buffer to each column.
  • Spun at max rpm for 30 seconds.
  • Put marked (CMV gel pur2 3/22) CMV in -20°C fridge in box labeled Cameron.
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