Haynes Lab:Notebook/Engineering PC-TFs/2013/09/12

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(Summary)
Current revision (00:19, 12 October 2013) (view source)
(September 12, 2013)
 
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==Summary==
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==September 12, 2013==
* Follow steps for gel electrophoresis.
* Follow steps for gel electrophoresis.
*  Orient top notch of gel electrophoresis apparatus. Place gel tray into larger tray.
*  Orient top notch of gel electrophoresis apparatus. Place gel tray into larger tray.
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| bgcolor=#cfcfcf | '''Reagent'''
| bgcolor=#cfcfcf | '''Reagent'''
| bgcolor=#cfcfcf | '''Volume'''
| bgcolor=#cfcfcf | '''Volume'''
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| rowspan="7" | [[Image:cmvgel1.jpg|150px| CMV and MV9 Stock digest]] [[Image:cmvgel1_cutout.jpg|150px|CMV and MV9 Stock Digest 3/18/2013]] <br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
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| rowspan="7" | [[Image:cmvgel2.jpg|250px| CMV and MV9 Stock digest]] [[Image:cmvgel1_cutout.jpg|250px|CMV and MV9 Stock Digest 3/18/2013]] <br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
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| DNA (plasmid) || 20.0 μL
| DNA (plasmid) || 20.0 μL
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| &nbsp; || 30 μL --> 37°C/ ~10 min.
| &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;" | Sequence
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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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| -1.17
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|-
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|2
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|CMV
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|2.767
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|-
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|3
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|CMV
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| -2.116
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|-
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|5
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|MV9
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|20.838
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|-
|}
|}

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September 12, 2013

  • Follow steps for 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 and MV9 Stock digest CMV and MV9 Stock Digest 3/18/2013
30 μL/lane, 1% agarose; Ladder
DNA (plasmid) 20.0 μL
SpeI 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
# Sequence ng/µL
1 CMV -1.17
2 CMV 2.767
3 CMV -2.116
5 MV9 20.838


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