James+F.+(Springer)

= = = Weeks 14 and 15 = = = = = =Virtual Screening (11/25 and 11/26):=



Table 2 – Best ranking list of the top 30 novel compounds. Compounds from the NIH_ClinCol3Ded library are shown in green. Compounds from the In-house library are shown in red. Compounds from the cb_306_3d library are shown in cyan. Lipinski’s Rule of 5 information, binding subscores from GOLD, formula, and chemical name are shown. Top 5 compounds that satisfy Lipinski’s Rule of 5 are highlighted in yellow. Compounds that do not satisfy Lipinski’s Rule of 5 are highlighted in red.





Table 1 – Best ranking list of 10 positive controls and 5 negative controls. Negative controls are the ZINC ligands and aspirin. Lipinski’s Rule of 5 information, rotatable bonds, binding subscores from GOLD chemical name, formula, and polar contacts are shown = =

__** Analysis: **__ The scores for the control ligand were decent. The highest score was a 71.6 and the lowest was 40.73. The positive controls did not necessarily perform better than the negative controls, with the top score actually being a negative control. Aspirin had the second to lowest score, which was expected as we had no reason to believe that Aspirin would bind well to the protein. There also did not appear to be a strong correlation between GOLD fitness score and the number of polar contacts. One would expect a compound with a greater number of polar contacts to have a higher fitness score, but this is not necessarily true in this case. For the novel compound screening, there were several scores that were high (with a 99.55 being the highest). In fact, the top 10 highest scores were all from the same library - NIH_ClinCol3Ded. Unfortunately, three very high scoring compounds from this library did not satisfy Lipinski’s Rule of 5 due to a high molecular weight or by having too many hydrogen bond donors or acceptors. After eliminating these compounds, the highest score was 92.09 from SAM001246657. This would currently be my best choice. The other 4 best choices have much lower gold scores at 82.82, 81.62, 81.1, and 79.63 respectively. Possible sources of error would be if I had incorrectly prepared the protein file in Hermes. Forgetting to remove waters or add hydrogens could impact binding affinity between the active site and the ligand. Another error would be if I had incorrectly prepared the control ligands during Ligand Prep with Maestro. An incorrectly protonated ligand or one with an incorrect force field may not result in accurate binding with the protein’s active site which would lead to skewed results. ___

= 120042014- Good job = = Weeks 11, 12 and 13 =

=PCR Squared (11/18 and 11/20):=





=Secondary PCR (11/11):=



=Primary PCR (11/10):=



Primary PCR was redone to obtain a better yield for future steps (2 uL of oligomix and 1 uL of Q5 polymerase was added instead of 1 uL and 0.5 uL, respectively). In figure 1, primary PCR was successful (shown by the streak in lane 3) by using NEB recommended guidelines with an annealing temperature of 62.5 degrees C. Secondary PCR was also successful (shown by the dark band in lane 9) in figure 2 using NEB recommended guidelines with an annealing temperature of 54.5 degrees C. PCR Squared was attempted two times shown in figures 3 and 4. Each one was unsuccessful and the same protocol for secondary PCR was followed. 4 dark bands in lanes 3-6 should have been present for both gels. A source of error would be poor mixing after creating the master mix for PCR Squared. Since 4 tubes need to be created, the solution must be split into 4 parts. However, if the parts are unevenly distributed in the master mix, certain tubes may have less or more reagents such as Q5 polymerase or dNTPs which would cause the reaction to fail. Another source of error would be spilling during pipetting. This would skew the concentrations of the reagents and could negatively impact the success of the experiment.

1162014- Great Job

= Weeks 8, 9 and 10 = =Secondary PCR (10/21, 10/29, 10/30, 10/31):=









=Primary PCR (10/21):= = = = = Figure 1 – Gel electrophoresis of lmptp after primary PCR (second attempt) with 1% agarose gel with TAE buffer and 0.5 uL of ethidium bromide. Lane 1 – skipped, lanes 2, 3, and 6 – other students’ samples, lane 4 – 5 uL of 1 kb DNA ladder, lane 5 – 12 uL of primary PCR sample of lmptp = = __Analysis:__ MY second attempt at primary PCR succeeded. A smear is visible on lane 5 in figure 1. 62 degrees C was used this time for the annealing temperature and NEB guidelines were followed and the middle number was used for when there was a range for time (ex - 25 seconds if the range was from 20-30 seconds). The smear present indicates that primary PCR has worked. For secondary PCR, all 4 attempts failed. No band was observed in lane 3 in figure 2. NEB guidelines with an annealing temperature of 62 degrees was used and the middle number was used for time when there was a range. On the second attempt (figure 3), no band was observed in lane 10. NEB guidelines with an annealing temperature of 62 degrees C and an annealing time of 25 seconds was used. For the third attempt (figure 4), no band was observed in lane 3. For this attempt, NEB guidelines with an annealing temperature of 62 degrees C with an annealing time of 20 seconds and an extension time of 40 seconds was followed. On the fourth atempt (figure 5), no band was observed in lane 3 and DNA ladder was accidently added twice in lanes 2 and 4. NEB guidelines with an annealing temperature of 54.5 degrees C was used and the middle number in he range was used for times. A source of error would be using the wrong annealing temperature. Failure to anneal would result in Q5 polymerase failing to proceed with DNA formation. Another error could be the annealing and extension times. Since annealing and extension are both temperature and time sensitive, a wrong time would result in either step to fail.

___

= Week 5&6 = 9232014- Good work, but you should add more pictures =Primary PCR (10/8):=





__Analysis:__ The first attempt at primary PCR failed. Two DNA ladders were accidently added in lanes 2 and 4 and 17 uL of sample was added in lane 3. No smear was observed on the gel. The instructions that were followed was the thermocycler cycling conditions. The second attempt at primary PCR also failed. The instructions followed for this attempt were the NEB recommended guidelines. The DNA ladder was observed in lane two and my sample was loaded in lane 7, but there was no smear present on lane 7. A source of error include using the wrong annealing temperature. The provided temperature of 58 degrees C is general and my melting point temperature for my oligo primers (minus 5 degrees Celsius) could be different than this. Failure to anneal would result in my primers not attaching to the DNA template and as a result, Q5 polymerase would fail to proceed with DNA formation. The next step would be to redo primary PCR at the temperature that is closer to my oligoprimer’s melting point minus 5 degrees.

=MIDI-Prep (10/2):=





__ Analysis: __ MIDI-prep was successfully completed and an average yield of 44.9 ng/uL (44.1 ng/uL and 45.7 ng/uL) of pNIC-Bsa4 was obtained. A source of error would be if I had accidently eluted DNA using buffers QF and TE into the waste flask and not into the conical tube. This would have resulted in a decrease in DNA concentration after nanodrop. Another source of error would be if I had accidently applied back pressure to the QIAprecipitator, which would have damaged the midi module’s membrane and result in me losing my DNA. The next step would be to proceed with cloning and use PCR to synthesize my gene of interest and insert it into pNIC-Bsa4. _

09232014- Outstanding work, keep up the good work. = Week 3&4 =

=PCR (9/18):=

__**Analysis:**__ My gel was mostly successful. The gel was expected to have just one band in a same location for lanes 2, 3 and 4. Lane 5 with sample D was not supposed to have a band because there was no DNA in that sample. There seems to be an error in loading sample A in lane 3 because there is not a band. This could have been caused by error during pipetting where not enough sample was added. Lanes 4-5 increase in intensity of color, which is expected because a higher concentration of sample should result in a darker band. Lane 6 (no DNA) did not have a band as expected.

=Restriction Enzyme Digest (9/8)=



=**__Analysis:__**= Results indicate that the experiment has (mostly) worked. Lane 4 with sample 1 (plasmid cut with EcoR1 restriction enzyme) had one strong, dark band. Lane 5 with sample 2 (plasmid cut with PvuII restriction enzyme) had 2 strong, dark bands as expected because PvuII makes 2 cuts. Lane 6 with sample 3 (plasmid cut with both EcoR1 and PvuII restriction enzymes) had 3 strong, dark bands as PvuII makes 2 cuts and EcoR1 makes 1 cut. Unfortunately, an error was probably made in lane 3. There does not seem to be very much uncut plasmid. There was probably less than 100 ng of plasmid added. As a result, a strong band is not present. This could have been because the plasmid was not pipetted correctly. As a result, the plasmid may have been inserted somewhere else on the gel.


 * Day 2 - Transformation (9/8)**

Forgot to take a picture of the flask after incubation.

==
 * Day 2 - Transformation (9/9)**

=**__Analysis:__**= From figure 1, we can see that the liquid is turbid. This indicates E. coli growth. After spinning down to remove the liquid, we have obtained pellets that can be used for purification in a future step. Pellets were stored in the 20 degrees Celsius refrigerator. ___ = Week 1&2 = Figure 3 – 10 uL mixture of E. Coli DH5 Alpha and pNIC-Bsa4 plasmid that was heat shocked for 45 seconds at 42 degrees Celsius and S.O.C. media that was spread with colirollers on LB+Kan agar plate

Figure 2 – 50 uL mixture of E. Coli DH5 Alpha and pNIC-Bsa4 plasmid that was heat shocked for 45 seconds at 42 degrees Celsius and S.O.C. media that was spread with colirollers on LB+Kan agar plate

__**Analysis:**__ Bacterial growth is present in both the 10 microliter and 50 microliter plate. As expected, the plate that contains the 50 microliter bacteria mixture (E. Coli DH5 Alpha) has more colonies. Although growth was observed, one can not determine if the pNIC-Bsa4 plasmid has been successfully transformed into the bacterial genome. The next step would be to extract a colony and grow it in a tube so we can over-express the protein. Plates from Aptamers were as instructed by mentors.

Figure 1 - Nanodrop measurement #2 using ND-1000 to determine the concentration of pGBR-22 sample. Unfortunately, we forgot to take a screenshot of the first measurement.

__**Analysis:**__ DNA absorbs light at a maximum wavelength of around 260 nm. This is consistent with the graph as there is a peak at 260 nm. 315.3 ng/uL of DNA was obtained from the pGBR-22 sample. Mentors have stated that this amount is sufficient. In addition, my 260/280 ratios (1.87 and 1.88) are close to the expected value of 1.8. This value indicates that the purity in relation to protein is satisfactory. The 260/230 values are 2.44 and 2.34. This indicates that the purity in relation to the other contaminants is satisfactory because both values are close to the expected value of 2.1.