Midi Prep Results and DNA Concentration

By far, the Midi Prep lab has been one of my most favorite and yet my longest lab to this day. I enjoyed seeing the visual changes and understanding the chemical reactions that were causing these changes. Some errors to note are:

– did not add isopropanol on Step 13, so I had to re-centrifuge the tube with isopropanol

-while in the centrifuge for the 2nd time on step 13, it was stopped constantly for 15 minutes due to trying to accommodate another researchers samples

-did not save any of the analytical samples for future testing

This is a graphical representation of the concentration of the DNA. Notice on the side in green, the concentration of the DNA is 474.3 ng/uL.

After the completion of the Midi Prep, I was able to use the Nanodrop to determine the concentration of my isolated DNA sample. In the end, I had about 474.3 ng/uL of pure DNA in my sample.

PyMOL Refresher Results

These are the results from the PyMOL refresher.

Fig 1: This is a picture of the NAP molecule (in red) in the active site of the 2H2Q active site. Notice the yellow dashed lines which represent the polar contacts between the substrate and the protein’s active site. There are several polar contacts which can suggest that the active site is highly polar and this is a hydrophilic substrate.

Fig 2: This picture shows the DU cofactor (in red) that is found in the 2H2Q protein. It is possible to say that this cofactor is slightly hydrophobic due to the small amount of polar contacts between the cofactor and the protein’s active site.

Fig 3: This picture shows the NAP substrate (in red) in the 3CL9 protein’s active site. The NAP substrate is a hydrophilic molecule and this can be seen based on the high number of polar contacts between it and the protein’s active site.

Fig 4: This picture shows the MTX substrate (in red) in the 3CL9 protein’s other active site. This molecule has hydrophilic tendencies which can be seen in the number of polar contacts it has with the active site of the protein.

Fig 5: This picture shows the polar contacts between the active site of 3CL9 and the UMP cofactor (in red). This cofactor, too, is hydrophilic because of the number of polar contacts between this molecule and the active site.

Fig 6: This picture shows the EDO cofactors (in red) in the 3CL9 protein. These molecules are highly hydrophobic because they are found inside of the protein. Also, there are not a lot of polar contacts between the cofactors and the protein.

Fig 7: This picture shows the chlorine atoms (in red) that are found within the 3CL9 protein complex. There are no polar contacts between the chlorine atoms and the 3CL9 protein.

Fig 8: This picture shows the superimposed structures of the 1U72 and 3CL9 protein proteins. The 3CL9 protein is in blue and the 1U72 protein is in red. The RMS value for this superimposition is 3.571.

Fig 9: This picture is of the protein 3HBB. The hydrophobic amino acids are in red, the polar amino acids are in blue, and the ionic amino acids are in yellow.

Fig 10: This picture shows the NAP substrates (in red) in the active sites of the 3HBB protein. These molecules are hydrophilic which can be seen through their large amounts of polar contacts between the NAP molecule and the protein.

Fig 11: This picture shows all of the substrates and cofactors found within the 3HBB protein complex. The following cofactors and substrates are: TMQ (in green) cofactor, the SO4 (in yellow) cofactor, the NAP substrates (in red), and the EDO (in pink) cofactor. None of the cofactors have shown any visible polar contacts.

Rought Draft of Research Report

The rough drafts for the research report can be uploaded to the Google Docs folder /Results&Image/ReportOnTarget

be sure to use the standard file name format

UTEID_date_ResearchReportRoughDraft.pdf    (please convert to PDF since it is easier for me to make comments

Dr. B

Week of 9/20-9/24

Finally some success! On Monday morning a sent in Sample 3 and 4 (both of which came from the same colony) to DNA sequencing. #3 reflected the appearance of the other samples and well as looked like cut pNIC-Bsa4 so I assumed it had failed. #4 looked slightly different but still did not look like the online simulation of the cut Zhang gene. When I got the results back, I blasted the forward and reverse sample 4 with Zhang and it came out as a perfect match. This was much better than I expected. Then, I had to blast #3 because if it turned out to not be Zhang, I would have a mixed colony which could cause problems in the future. It also matched much to my surprise. Then I found the colony on my original plate that both samples came from and grew up 2 flasks of 80ml each of this colony. It grew over night and left me with large pellets after being spun down. Next week I’m going to send a sample to DNA sequencing again because my plate had overgrown a bit and the colony might have been tainted. Hopefully I will be moving forward  next week.

On a different note, if anyone can tell me how to embed images, I would really appreciate that. Hipe you all have a fun weekend!