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

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(October 11, 2013)
(October 11, 2013)
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|   || 30 μL --> 37°C/ ~10 min.
 
|   || 30 μL --> 37°C/ ~10 min.
 
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''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"
 
{|border="1" cellpadding="5" cellspacing="0" align="left"
 
|-
 
|-
 
! scope="col" style="background:#efefef;" | #
 
! scope="col" style="background:#efefef;" | #
! scope="col" style="background:#efefef;" | Sequence
+
! scope="col" style="background:#efefef;" | Part
 
! scope="col" style="background:#efefef;" | ng/µL
 
! scope="col" style="background:#efefef;" | ng/µL
 
|-
 
|-
 
|1
 
|1
 
|CMV
 
|CMV
| -1.17
+
|0.851
 
|-
 
|-
 
|2
 
|2
 
|CMV
 
|CMV
|2.767
+
|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