Difference between revisions of "Haynes Lab:Notebook/Engineering PC-TFs/2013/10/11"

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(Autocreate 2013/10/11 Entry for Haynes_Lab:Notebook/Engineering_PC-TFs)
 
(October 11, 2013)
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==Summary==
+
==October 11, 2013==
*  
+
'''Restriction Digest'''
 +
 
 +
''Gel Electrophoresis''
 +
 
 +
*  Orient top notch of gel electrophoresis apparatus. Place gel tray into larger tray.
 +
*  Fill gel flask with up to 60 ml of TA buffer.
 +
*  Create 1% gel by putting .6 grams of agarose into flask.
 +
*  Microwave agarose solution for 40 seconds
 +
*  Using hot gloves, grab flask out of microwave and swirl flask vigorously. Place back in microwave for 40 more seconds.
 +
*  When flask is taken out of microwave, make sure that the agarose is completed dissolved.
 +
*  Set up wells on gel electrophoresis apparatus. The mold for the first digest is B-10. Use the thick "teeth" and slip in the notches.
 +
*  Add EtBr (ethidium bromide) to the agarose gel. There should be 1 microliter of EtBr per 10ml of agarose gel. Therefore, 6 µL.
 +
*  Pour gel into tray.
 +
*  Wash the agarose gel flask.
 +
* Run gel for 45 min at 100 V. Check gel under UV light.
 +
 
 +
{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
 +
|- valign="top"
 +
| bgcolor=#cfcfcf | '''Reagent'''
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| bgcolor=#cfcfcf | '''Volume'''
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| rowspan="7" | [[Image:abc12.jpg|250px| CMV Stock digest 10/11/2013]] [[Image:whyohwhy.jpg|250px|CMV Stock Digest 10/11/2013]] <br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
 +
|-
 +
| DNA (plasmid) || 20.0 μL
 +
|-
 +
| XbaI|| 1.0 μL
 +
|-
 +
| PstI|| 1.0 μL
 +
|-
 +
| Green 10x FastDigest buffer || 3.0 μL
 +
|-
 +
| dH<sub>2</sub>O || 5 μL
 +
|-
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| &nbsp; || 30 μL --> 37°C/ ~10 min.
 +
|}
 +
 
 +
''Zymoclean<sup>TM</sup> Gel DNA Recovery Kit''
 +
 
 +
*Add 3 volumes of ADB Buffer to each volume of gel (600 μL).
 +
*Incubate at 55°C for 10 minutes.
 +
*Centrifuge at ≥ 10,000 x g for 30 seconds. Discard flow-through.
 +
*Add 200 μL of DNA Wash Buffer to the column and centrifuge for 30 seconds. Repeat wash step.
 +
*Place Zymoclean Column into a new 1.5 mL tube. Add 20μL of dH<sub>2</sub>O to elute DNA
 +
 
 +
{|border="1" cellpadding="5" cellspacing="0" align="left"
 +
|-
 +
! scope="col" style="background:#efefef;" | #
 +
! scope="col" style="background:#efefef;" | Part
 +
! scope="col" style="background:#efefef;" | ng/µL
 +
|-
 +
|1
 +
|CMV
 +
|0.851
 +
|-
 +
|2
 +
|CMV
 +
|15.535
 +
|}
  
  

Revision as of 20:18, 11 October 2013


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October 11, 2013

Restriction Digest

Gel Electrophoresis

  • Orient top notch of gel electrophoresis apparatus. Place gel tray into larger tray.
  • Fill gel flask with up to 60 ml of TA buffer.
  • Create 1% gel by putting .6 grams of agarose into flask.
  • Microwave agarose solution for 40 seconds
  • Using hot gloves, grab flask out of microwave and swirl flask vigorously. Place back in microwave for 40 more seconds.
  • When flask is taken out of microwave, make sure that the agarose is completed dissolved.
  • Set up wells on gel electrophoresis apparatus. The mold for the first digest is B-10. Use the thick "teeth" and slip in the notches.
  • Add EtBr (ethidium bromide) to the agarose gel. There should be 1 microliter of EtBr per 10ml of agarose gel. Therefore, 6 µL.
  • Pour gel into tray.
  • Wash the agarose gel flask.
  • Run gel for 45 min at 100 V. Check gel under UV light.
Reagent Volume CMV Stock digest 10/11/2013 CMV Stock Digest 10/11/2013
30 μL/lane, 1% agarose; Ladder
DNA (plasmid) 20.0 μL
XbaI 1.0 μL
PstI 1.0 μL
Green 10x FastDigest buffer 3.0 μL
dH2O 5 μL
  30 μL --> 37°C/ ~10 min.

ZymocleanTM Gel DNA Recovery Kit

  • Add 3 volumes of ADB Buffer to each volume of gel (600 μL).
  • Incubate at 55°C for 10 minutes.
  • Centrifuge at ≥ 10,000 x g for 30 seconds. Discard flow-through.
  • Add 200 μL of DNA Wash Buffer to the column and centrifuge for 30 seconds. Repeat wash step.
  • Place Zymoclean Column into a new 1.5 mL tube. Add 20μL of dH2O to elute DNA
# Part ng/µL
1 CMV 0.851
2 CMV 15.535