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)
Current revision (23:18, 11 October 2013) (view source)
(October 11, 2013)
 
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==Summary==
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==October 11, 2013==
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*  
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'''Restriction Digest'''
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''Gel Electrophoresis''
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* Orient top notch of gel electrophoresis apparatus. Place gel tray into larger tray.
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*  Fill gel flask with up to 60 ml of TA buffer.
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*  Create 1% gel by putting .6 grams of agarose into flask.
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*  Microwave agarose solution for 40 seconds
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*  Using hot gloves, grab flask out of microwave and swirl flask vigorously. Place back in microwave for 40 more seconds.
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*  When flask is taken out of microwave, make sure that the agarose is completed dissolved.
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*  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.
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*  Add EtBr (ethidium bromide) to the agarose gel. There should be 1 microliter of EtBr per 10ml of agarose gel. Therefore, 6 µL.
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*  Pour gel into tray.
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*  Wash the agarose gel flask.
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* Run gel for 45 min at 100 V. Check gel under UV light.
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{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
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|- valign="top"
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| 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]
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|-
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| DNA (plasmid) || 20.0 μL
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|-
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| XbaI|| 1.0 μL
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|-
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| PstI|| 1.0 μL
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|-
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| Green 10x FastDigest buffer || 3.0 μL
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|-
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| dH<sub>2</sub>O || 5 μL
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|-
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| &nbsp; || 30 μL --> 37°C/ ~10 min.
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|}
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''Zymoclean<sup>TM</sup> Gel DNA Recovery Kit''
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*Add 3 volumes of ADB Buffer to each volume of gel (600 μL).
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*Incubate at 55°C for 10 minutes.
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*Centrifuge at ≥ 10,000 x g for 30 seconds. Discard flow-through.
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*Add 200 μL of DNA Wash Buffer to the column and centrifuge for 30 seconds. Repeat wash step.
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*Place Zymoclean Column into a new 1.5 mL tube. Add 20μL of dH<sub>2</sub>O to elute DNA
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{|border="1" cellpadding="5" cellspacing="0" align="left"
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|-
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! scope="col" style="background:#efefef;" | #
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! scope="col" style="background:#efefef;" | Part
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! scope="col" style="background:#efefef;" | ng/µL
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|-
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|1
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|CMV
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|0.851
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|-
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|2
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|CMV
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|15.535
 +
|}

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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



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