User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/11/06: Difference between revisions
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* 40μL of E.coli cells was added into a 1mL microcentrifuge tube, 10μL of ADA E34K plasmid was added into bacteria without mix. | * 40μL of E.coli cells was added into a 1mL microcentrifuge tube, 10μL of ADA E34K plasmid was added into bacteria without mix. | ||
* The culture was placed on heat block for heat shock for 30 seconds. After heat shock, the culture was placed back on ice. | * The culture was placed on heat block for heat shock for 30 seconds. After heat shock, the culture was placed back on ice. | ||
* 250μL of | * 250μL of SOC media obtained from New England Biolabs® was added to bacteria culture. The microcentrifuge tube was taped and attached to a small test tube. Sample was placed in orbital shaker under the following conditions: | ||
225rpm | 225rpm | ||
37°C | 37°C | ||
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==Procedure for UV-vis on Au/Lysozyme samples== | ==Procedure for UV-vis on Au/Lysozyme samples== | ||
* Au/Lysozyme samples made on [[User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/10/31|2012/10/31]] were used to take spectrum of all samples via UV-vis spectrometer. The below ratios of Au/Lysozyme sample were run: | |||
20 - 30 - 40 - 50 - 60 - 70 - 80 - 120 - 130 | |||
* The appearance of Au/Lysozyme samples were noted and displayed in results section below. | |||
* The UV-vis spectrometer was set to spectrum method and wavelength from 200nm to 800nm was ran for all samples. | |||
* Samples were run with distilled water as baseline standards. 2mL of samples were placed in quartz cuvette for each sample run. | |||
* Absorbance at each wavelength for the above ratios were recorded and plotted on graph, shown in results section below. | |||
==Results for UV-vis on Au/Lysozyme samples== | ==Results for UV-vis on Au/Lysozyme samples== | ||
* The Au/Lysozyme solutions have various appearances based on the ratio of gold to lysozyme. For Au/Lysozyme with mole ratio of 20 to 60, the supernatant appeared purple in color and clear. No fibers were observed. For Au/Lysozyme with mole ratio of 70 to 120, the supernatant appeared clear with purple fibers formed and collected at the bottom of test tube. | |||
* Detailed observation for each mole ratio of Au/Lysozyme were recorded in table below: | |||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Au/Lysozyme ratio''' | | align="center" style="background:#f0f0f0;"|'''Au/Lysozyme ratio''' | ||
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|- | |- | ||
| 80||Clear supernatant, purple fibers formed aggregating at the bottom of test tube. Some fibers were floating in supernatant. | | 80||Clear supernatant, purple fibers formed aggregating at the bottom of test tube. Some fibers were floating in supernatant. | ||
|- | |- | ||
| 120||Clear supernatant, purple fibers formed aggregating at the bottom of test tube. Some fibers were floating in supernatant. | | 120||Clear supernatant, purple fibers formed aggregating at the bottom of test tube. Some fibers were floating in supernatant. | ||
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| 130||Clear supernatant, purple fibers formed aggregating at the bottom of test tube | | 130||Clear supernatant, purple fibers formed aggregating at the bottom of test tube | ||
|} | |} | ||
* The supernatant were used to ran UV-vis spectrometry. A graph with absorbance versus wavelength from 200nm to 800nm were plotted and shown below: | |||
[[Image:AuNP-Lysozyme_Absorbance.jpg|600px]] | |||
* From the graph above, it is shown that Au/Lysozyme with mole ratio of 40 yielded the greatest absorbance, and hence indicated the greatest number of gold nanoparticles in solution. Followed by 40 is mole ratio of 50, then mole ratio of 60 that yields decreasing concentration of gold nanoparticles in solution. This indicates that the concentration of gold nanoparticles decrease after the maximum mole ratio of 40. | |||
* It was expected for mole ratio of 130 Au/Lysozyme sample to have an absorbance around 0.00. But according to graph, the absorbance for sample came out to be 0.04. This might caused by accidental resuspension of purple fibers into supernatant when withdrawing the supernatant for sampling. | |||
* The peak at wavelength 525nm was looked at separately based on [http://pubs.acs.org/doi/abs/10.1021/jp110296y Bakshi, et al], which stated that gold nanoparticles can be indicated through a peak at 530nm when gold nanoparticles are present in samples. A table listing absorbance at 530nm for each mole ratios of Au/Lysozyme is shown below: | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Au/Lysozyme ratio''' | |||
| align="center" style="background:#f0f0f0;"|'''Absorbance at 525nm''' | |||
|- | |||
| 20||0.056 | |||
|- | |||
| 30||0.077 | |||
|- | |||
| 40||0.269 | |||
|- | |||
| 50||0.235 | |||
|- | |||
| 60||0.193 | |||
|- | |||
| 70||0.036 | |||
|- | |||
| 80||0.036 | |||
|- | |||
| 120||0.048 | |||
|- | |||
| 130||0.083 | |||
|} | |||
* Based on the table, a graph of absorbance versus increasing mole ratios of Au/Lysozymes were made to better depict relationship between concentration of gold nanoparticles in supernatant and mole ratios of Au/Lysozymes. The graph is shown below: | |||
[[Image:KW.png|350px]] | |||
* From the graph above, it can be shown that the concentration of gold nanoparticles in supernatant increases as the mole ratio of Au/Lysozyme goes from 20 to 40. The concentration of gold nanoparticles peaks at mole ratio of 40, then decreases from there on as ratio of Au/Lysozyme increases further. It seems like that the concentration of gold nanoparticles in supernatant increases from mole ratio of 120 to 130, but it might be caused by accident resuspension of fibers when absorbance of sample 130 Au/Lysozyme was taken. | |||
* The 40 mole ratio of Au/Lysozyme can be further investigated from 20 to 50 to determine an exact ratio of gold to lysozyme that encouraged protein aggregation. | |||
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Revision as of 11:55, 25 November 2012
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Purpose
Procedure for Protein Extraction and Purification from BL21(DE3) Cells
Speed: 18,000rpm Temperature: 4°C Time elapsed: 2 hours Rotor used:_____
Result for Protein Extraction and Purification from BL21(DE3) Cells
Procedure for E.coli cell Transformation
225rpm 37°C 1 hour
Following reagents were weighted out: 20g of LB Agar powder from Teknova® 5g of tryptone from Teknova® 2.5g of yeast extract from BD® 5g of NaCl from Fisher Scientific® 7.5g agar from BD® Above were placed into a 800mL Erlenmeyer flask. 500mL distilled water was added to reagents. The solution was autoclaved under liquid cycle (see 2012/11/03 for protocol for liquid cycle) After cooling to ~50°C, 50mg ampicillin was poured into autoclaved solution, swirled to mix Under sterile condition, the solution was poured into separate petri dish when temperature reaches ~40°C Plates were left under sterile condition to solidify
Results for E.coli cell Transformation
Procedure for UV-vis on Au/Lysozyme samples
20 - 30 - 40 - 50 - 60 - 70 - 80 - 120 - 130
Results for UV-vis on Au/Lysozyme samples
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