User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/11/06: Difference between revisions
From OpenWetWare
Keyun Wang (talk | contribs) |
Keyun Wang (talk | contribs) No edit summary |
||
Line 8: | Line 8: | ||
==Purpose== | ==Purpose== | ||
* Protein was extracted from transfected BL21(DE3) E.coli cells and adenosine deaminase was purified via column chromatography. | * Protein was extracted from transfected BL21(DE3) E.coli cells and adenosine deaminase was purified via column chromatography. | ||
* Adenosine deaminase plasmid with the mutation E34K was transformed into | * Adenosine deaminase plasmid with the mutation E34K was transformed into DH5α-T1 E.coli cells with a second attempt. | ||
* UV-vis spectroscopy was used to analyze Au/Lysozyme solutions made on [[User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/10/31|2012/10/31]] to measure absorbance of supernatant of Au/Lysozyme samples | * UV-vis spectroscopy was used to analyze Au/Lysozyme solutions made on [[User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/10/31|2012/10/31]] to measure absorbance of supernatant of Au/Lysozyme samples | ||
Line 37: | Line 37: | ||
* Because the UV-vis spectrum from FPLC was not saved, data cannot be displayed. However, there was a clear peak at 280nm that indicates presence of ADA protein. | * Because the UV-vis spectrum from FPLC was not saved, data cannot be displayed. However, there was a clear peak at 280nm that indicates presence of ADA protein. | ||
* The absorbance peak at 280nm was the highest in fraction 2 of elution, fraction 2 was suspected to have the highest concentration of ADA proteins. Furthermore, it indicated that ADA protein started to unbind from the column via competitive binding with imidazole in fraction 1. Both fraction 1 and 3 also yielded an absorbance peak at 280nm but not as high as the ones from fraction 2. As a result, fraction 1 and 3 were also collected for maximum yield of ADA proteins in the same falcon tube. | * The absorbance peak at 280nm was the highest in fraction 2 of elution, fraction 2 was suspected to have the highest concentration of ADA proteins. Furthermore, it indicated that ADA protein started to unbind from the column via competitive binding with imidazole in fraction 1. Both fraction 1 and 3 also yielded an absorbance peak at 280nm but not as high as the ones from fraction 2. As a result, fraction 1 and 3 were also collected for maximum yield of ADA proteins in the same falcon tube. | ||
==Procedure for E.coli cell Transformation== | |||
* DH5α-T1 Competent E. coli cells was obtained from -80°C freezer and was thawed on ice bath after 30 minutes. | |||
* PCR products with the mutation E34K in ADA plasmid strand obtained from [[User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/10/16|2012/10/16]] was thawed on ice bath. | |||
* 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. | |||
* 250μL of LB media 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 | |||
37°C | |||
1 hour | |||
* Cell culture was taken out of shaker, and plated on LB agar plates made with LB, agar, and ampicillin. The LB plates were made in the following way: | |||
2g of LB Agar powder from Fisher Scientific was weighted out. | |||
==Results for E.coli cell Transformation== | |||
* After 15 hours in incubator, the plates appeared to have no colonies. | |||
* This indicates that the transformation did not work. This procedure is on hold for trouble shooting. | |||
{| {{table}} | {| {{table}} |
Revision as of 20:59, 24 November 2012
Experimental Biological Chemistry | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | ||||||||||||||||||||||
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
2g of LB Agar powder from Fisher Scientific was weighted out. Results for E.coli cell Transformation
|