User:Michael F. Nagle/Notebook/Chem 571/2012/10/17

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(Autocreate 2012/10/17 Entry for User:Michael_F._Nagle/Notebook/Chem_571)
Current revision (03:23, 7 December 2012) (view source)
 
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==Entry title==
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==Objectives==
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* Insert content here...
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*Compare molar mass of plasmids via electrophoresis
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*Prepare new sets of 60-170 [Au/BSA] and [Au/Lysozyme]
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*Analyze chemiluminescence of HRP assay with luminol using modified UV/Vis instrument
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==Electrophoresis==
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*5 μL of the plasmid mutated last week was placed in an autoclaved microcentrifuge tube.  
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*1 μL 6x blue gel loading dye was added to this.  
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*1 μL DPN1 was added to the other tube containing 45 μL plasmid solution.  
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*This solution was placed on a VWR heat block at 37°C.
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*Agarose gel was prepared by adding 1.23g agarose to 35mL TAE buffer and heating via microwave for 35 seconds.
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*The agarose was poured into a Horizon 58 electrophoresis tray.
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*Once the gel had solidified after 30 minutes, the comb was removed.
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*The tray was filled with TAE buffer.
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*The plasmid and dye were added to wells.
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*Electrophoresis was run and the instrument was powered with a Bio-Rad Powerpac 200.
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*Upon completion of electrophoresis, 20μL 12.3g/mL ethidium bromide was added and slowly mixed with a VWR shaker.
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*The gel was viewed under UV light.
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==Chemiluminescence==
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*Because the fluorimeter was not working,[[User:Mary_Mendoza/Notebook/CHEM_571_Experimental_Biological_Chemistry_I/2012/10/17|Mary Mendoza]] tried to measure chemiluminescence of an HRP assay with luminol as prepared [[User:Michael_F._Nagle/Notebook/Chem_571/2012/10/16|yesterday]] using a UV/Vis instrument with the source lamp blocked, set to measuring energy with a slit width of 5nm. There was no signal.
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==Nucleation of AuNPs with BSA and Lysozyme==
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*[[User:Mary_Mendoza/Notebook/CHEM_571_Experimental_Biological_Chemistry_I/2012/10/17|Mary Mendoza]] prepared lysozyme and gold stock solutions.
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* Solutions for both BSA and lysozyme and HAuCl<sub>4</sub> were both prepared at the same mole ratios that [[User:Michael_F._Nagle/Notebook/Chem_571/2012/08/29|have previously been used with BSA]]. Since the same BSA stock was used, and the lysozyme stock was prepared at the same concentration, the volumes added to each tube did not change.
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**Both sets of solutions were capped with foil and heated at 85<sup>o</sup>C for 4 hours.
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==Results and Discussion==
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*Besides the DNA ladder, nothing moved down the agarose gel in electrophoresis. This may be due to a problem with the samples, so they should be recreated before attempting electrophoresis again.
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Objectives

  • Compare molar mass of plasmids via electrophoresis
  • Prepare new sets of 60-170 [Au/BSA] and [Au/Lysozyme]
  • Analyze chemiluminescence of HRP assay with luminol using modified UV/Vis instrument

Electrophoresis

  • 5 μL of the plasmid mutated last week was placed in an autoclaved microcentrifuge tube.
  • 1 μL 6x blue gel loading dye was added to this.
  • 1 μL DPN1 was added to the other tube containing 45 μL plasmid solution.
  • This solution was placed on a VWR heat block at 37°C.
  • Agarose gel was prepared by adding 1.23g agarose to 35mL TAE buffer and heating via microwave for 35 seconds.
  • The agarose was poured into a Horizon 58 electrophoresis tray.
  • Once the gel had solidified after 30 minutes, the comb was removed.
  • The tray was filled with TAE buffer.
  • The plasmid and dye were added to wells.
  • Electrophoresis was run and the instrument was powered with a Bio-Rad Powerpac 200.
  • Upon completion of electrophoresis, 20μL 12.3g/mL ethidium bromide was added and slowly mixed with a VWR shaker.
  • The gel was viewed under UV light.

Chemiluminescence

  • Because the fluorimeter was not working,Mary Mendoza tried to measure chemiluminescence of an HRP assay with luminol as prepared yesterday using a UV/Vis instrument with the source lamp blocked, set to measuring energy with a slit width of 5nm. There was no signal.

Nucleation of AuNPs with BSA and Lysozyme

  • Mary Mendoza prepared lysozyme and gold stock solutions.
  • Solutions for both BSA and lysozyme and HAuCl4 were both prepared at the same mole ratios that have previously been used with BSA. Since the same BSA stock was used, and the lysozyme stock was prepared at the same concentration, the volumes added to each tube did not change.
    • Both sets of solutions were capped with foil and heated at 85oC for 4 hours.

Results and Discussion

  • Besides the DNA ladder, nothing moved down the agarose gel in electrophoresis. This may be due to a problem with the samples, so they should be recreated before attempting electrophoresis again.


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