Haynes Lab:Notebook/Engineering PC-TFs/2013/09/26

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(Summary)
Current revision (15:22, 28 September 2013) (view source)
(September 26, 2013)
 
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==Summary==
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==September  26, 2013==
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''Restriction Digest''
*Perform restriction digest for CMV promoter
*Perform restriction digest for CMV promoter
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| PstI|| 1.0 μL
| PstI|| 1.0 μL
|-
|-
-
| Green 10x FastDigest buffer || 3.0 μL
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| Green 10x FastDigest buffer || 0μL
|-
|-
-
| dH<sub>2</sub>O || 15 μL  
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| dH<sub>2</sub>O || 18 μL  
|-
|-
| &nbsp; || 30 μL --> 37°C/ ~10 min.  
| &nbsp; || 30 μL --> 37°C/ ~10 min.  
|}
|}
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''PEG/MgCl<sub>2</sub> Precipitation''
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* This procedure was adapated from [http://openwetware.org/wiki/Protocol_Size_selective_DNA_precipitation_by_PEG/MgCl2 Size selective DNA precipitation]
*Measure the following into 1.5 mL tubes.
*Measure the following into 1.5 mL tubes.
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| Total || 100 μL || 100 μL|| 100 μL|| 100 μL|| 100 μL
| Total || 100 μL || 100 μL|| 100 μL|| 100 μL|| 100 μL
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*Centrifuge  for 15 minutes at 10,000g.
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''DNA Clean & Concentrator''
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* Although there was no pellet, ran a DNA Clean & Concentrator on samples.
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**Add 2 volumes of DNA Binder Buffer to each DNA sample (200 μL).
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**Place mixture in spin column into 2 ml collection tube.
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**Centrifuge at full speed for 30 seconds.
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**Add 20 μL of DNA Wash Buffer and centrifuge for 30 seconds. Repeat this step.
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**Place Zymo-Spin Column into a new 1.5 ml tube. Add 20 μL  of water directly to column matrix and spin to elute DNA.
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''Gel Electrophoresis''
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* Follow steps for 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.
 +
*  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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[[Image:hiddendragonsofthetemple.jpg|250px|CMV PEG/MgCl<sub>2</sub> Precipitation 9/26/2013]]
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Higher molecular mass DNA precipitates at lower PEG concentrations than lower molecular mass DNA, according to [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC342844/ Lis and Schleif 1975, NAR]. I kept DNA from the supernatant and discarded the precipitated DNA.
 +
 +
Therefore, I should see larger bands at 2% PEG and smaller bands at 15% PEG.

Current revision



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September 26, 2013

Restriction Digest

  • Perform restriction digest for CMV promoter
Reagent Volume
DNA (plasmid) 10 μL
XbaI 1.0 μL
PstI 1.0 μL
Green 10x FastDigest buffer 0μL
dH2O 18 μL
  30 μL --> 37°C/ ~10 min.

PEG/MgCl2 Precipitation

  • Measure the following into 1.5 mL tubes.
Reagent 15% 10% 7% 5% 2%
30% PEG 50 μL 33 μL 23 μL 17 μL 7 μL
DNA 15μL15 μL15 μL15 μL15 μL
dH2O 35 μL52 μL62 μL68 μL78 μL
Total 100 μL 100 μL 100 μL 100 μL 100 μL
  • Centrifuge for 15 minutes at 10,000g.

DNA Clean & Concentrator

  • Although there was no pellet, ran a DNA Clean & Concentrator on samples.
    • Add 2 volumes of DNA Binder Buffer to each DNA sample (200 μL).
    • Place mixture in spin column into 2 ml collection tube.
    • Centrifuge at full speed for 30 seconds.
    • Add 20 μL of DNA Wash Buffer and centrifuge for 30 seconds. Repeat this step.
    • Place Zymo-Spin Column into a new 1.5 ml tube. Add 20 μL of water directly to column matrix and spin to elute DNA.

Gel Electrophoresis

  • 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.

CMV PEG/MgCl2 Precipitation 9/26/2013

Higher molecular mass DNA precipitates at lower PEG concentrations than lower molecular mass DNA, according to Lis and Schleif 1975, NAR. I kept DNA from the supernatant and discarded the precipitated DNA.

Therefore, I should see larger bands at 2% PEG and smaller bands at 15% PEG.



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