Rishi+D.

12-8-12 Finished up research paper, updated Wikispaces, and updated journal. Enzyme assays were unsuccessful, thus inhibition assays were not conducted. However, i did observe other researcher's results and procedures on conducting the assays. ****Enzyme assay results from Suman A. were cited and stated in research paper. **In addition, I gave reasons to what the graph was supposed to look like.** Further details in research paper and lab journal.

12-6-12 Cleaned up lab by trashing tubes that were not necessary and removing tape from my box. I ran the eyewash station for 10 minutes and filled eyewash bottles with clean water. Lastly, I removed tape from the floor of the lab and removed "BioBrick" labels from drawers.

12-6-12 Virtual Screening Run 2 Results (Top 10 Ligands):

Homology model (Swiss-Model Workspace (2ie4C) Template from PDB (//L.// major)

12-5-12 Enzyme assay with FtHap; it was unsuccessful due to many reasons. 1. Bad technique with pipetting solution 2. The protein was left in the water bath for too long (incubation) 3. The protein is just not active (protein degraded, denatured) The values (absorbance) were too small to consider using for inhibition. During the collection of absorbance, the samples got mixed up. Also there isn't a linear function as the concentrations of enzyme increased. Also, one of the samples was thrown out because it was skewed from the rest of the data.

12-4-12 Conducted enzyme assays with Paul and Ling. Results are still being gathered. Hopefully, inhibition can be started with the surrogate FtHap.

11-30-12 Thermo Scientific: (Red=70kDa, Green=10kDa) Gel Results: __#|Protein__ Characterization SDS-PAGE Lane 1: Skipped Lane 2: 10-170kDa Protein Ladder Lane 3: Sample 1, Cell lysate after induction Lane 4: Sample 2, Soluble fraction Lane 5: Sample 3, Flow through Lane 6: Sample 4, Wash Lane 7: Elution 1 from Trial 1 (Tube A) Lane 8: Elution 2 from Trial 1 (Tube B) Lane 9-10: Skipped
 * Some of Elution 2 spilled into Lane 9 causing faded bands. Also, Cell Lysate before induction was not inserted into Lane because not available.**

Nanodrop Results after __#|Protein Purification__ (Surrogate Enzyme FtHap):

Elution 1 (Concentration of 1.40 mg/mL): Tube A from Trial 1

Elution 2 (Concentration of 0.11 mg/mL): Tube B from Trial 1

Elution 1 (Concentration of 1.08 mg/mL): Tube C from Trial 2

Elution 2 (Concentration of 0.10 mg/mL): Tube D from Trial 2

11-28-12 Worked on __#|protein__ characterization and have results that will be posted Friday. The gel was seen to produce bands and nanodrop results showed concentrations after __#|protein purification__ at a wavelength of 280nm.

==11-26-12 __**Dr. B, I posted this on Monday. (I assumed you were going to check wikispaces on Monday and not Sunday for Wiki check 11. I have a zero on blackboard because of this.) I do not know if my grade can be changed. However, I did win the journal club questions for one free wikispace check if that suffices. "Freebee from Journal Club Victory."**__== Sonification was completed before the Thanksgiving break. Conical tubes are placed in the -80 __#|degree__ Freezer.

Good - Dr. B 11/19/12 11-16-12 Results shown: Pellet A protein: 3.84g Pellet B protein: 4.33g Pellet C protein: 3.33g Pellet D protein: 3.54g
 * Different masses of protein pellet may have resulted by agitation of the centrifuge bottles when spin-down was complete, and/or pouring out the supernatant quickly resulting in some loss of protein.**


 * 11-15-12**
 * Paul, Ling, and I expressed protein from FtHAP. Protein pellets A and B were lysed and placed together in a 15ml conical vial, and the same went for Protein pellets C and D into another 15 ml conical vial. Protein was stored in the -80 __#|degrees__ Celsius freezer.**


 * 11-14-12**
 * Protein expression of FtHAP will take place now. DNA sequencing results were incorrect and needed to be resubmitted. Due to time, protein expression will be conducted by Paul, Ling, and me.**


 * 11-9-12**
 * Nanodrop Results of Sections 2-9 are shown in lab notebook. Concentrations were relatively weak due to improper technique or actual growth of bacteria taking place. Further errors are stated in lab notebook as well.**


 * Master plate shown below. Sections 2-9 extracted DNA has been submitted to DNA sequencing. //Leishmania// __#|forward__ primer has been included with the extracted DNA.**


 * 11-7-12**
 * Working with Paul, there are roughly 60 colonies produced on this plate. A master plate will take place for __#|next__ time and will be submitted to DNA sequencing.**


 * 11-2-12**
 * Gel Results are to follow by Paul. He will post the image.**
 * Nanodrop Results of pNIC-Bsa4**


 * Nanodrop Results of PCR^2 after PCR Clean-up**


 * 10-26-12**
 * PCR^2 Results:**
 * Lane 1: Skipped**
 * Lane 2: 100bp ladder**
 * Lane 3-6: PCR^2 Product**
 * Lane 7-10: Skipped**


 * PCR __#|Cleanup__ and Nanodrop will follow __#|next__ time as well as Cloning.**


 * 10-24-12**
 * Target has been switched to** Serine-threonine protein phosphatase, putative (Leishmania major)**. Cloning was not successful with //T. cruzi//. PCR^2 will take place with Paul's secondary PCR. Target page will be read to understand new disease.**


 * 102112- Rishi, ok good results from Virtual. Not sure what is up with the wet lab stuff. You may want to make __#|your__ own batch of pNIC-Bsa4. -- Dr. B**


 * 10-19-12**
 * pNIC-Bsa4 may have been contaminated when observing my gel. For next time, I will use a new concentration of pNIC-Bsa4 from someone else. PCR squared will be conducted again only if my original PCR squared does not fit in the accepting vector once results are observed.**


 * 10-17-12**
 * pNIC-Bsa4 Accepting Vector Gel Results:**
 * Lane 1: Skipped**
 * Lane 2: 100bp ladder**
 * Lane 3: Ruifei's PCR**
 * Lane 4: Ruifei's PCR**
 * Lane 5: Skipped**
 * Lane 6: Skipped**
 * Lane 7: Accepting Vector (failure)**
 * Lane 8-10: Skipped**


 * Unfortunately, my Gel Results failed again. I incubated my product for 3 hours at 37 degrees Celsius. My plasmid did not cut. Thus, I will have to redo experiment.**


 * 10-15-12**
 * Virtual Drug Screening ligands in Pymol have been printed and shown with polar contacts.**

//--Finished 2nd Run for Virtual Drug Screening Refresher.//
 * 10-13-12**
 * 101612 - rishi- you can show the results table of a few of your top hits here as 'results' for your Wikiposting. -- Dr. B**
 * 10-12-12**
 * I worked on Virtual Drug Screening Refresher Protocol and will perform Gel Electrophoresis next time.**
 * Nanodrop of pNIC-Bsa4 Accepting Vector**
 * -This result is much better than my last product as the concentration is 26ng/uL. Protocol will continue next time.**


 * 10-11-12**
 * I went ahead and incubated my pNIC-Bsa4 Accepting Vector in 37 degrees Celsius Water. Also, I worked on Virtual Drug Screening Protocol.**


 * 10-10-12**
 * Virtual Drug Screening protocol continued. I also combined the reagents to perform pNIC-Bsa4 preparation. As far as the PCR squared, reagents were combined without the polymerase and stored in freezer for next time.**

//Lab notebook gives reason for no gel but did not post on wikispaces. My gel tore when taken out of the buffer. An image was not taken because of this. I will redo the protocol. Also, PCR squared will be conducted again.//
 * 10-5-12**
 * 100912 - Rishi - Can you show your PCR images too. We have new T4 DNA Poly that may work better for you cloning (new dGTP and new dCTP also) - Dr. B**

Technique may have been the issue that caused my curve to be abnormal. I will conduct this part of the experiment again to obtain proper results.
 * Nanodrop (using Elution Buffer) pNIC-Bsa4**

10-3-12 I went ahead and started pNIC-BSA4 protocol. As of right now, my product is in incubation at 37 degrees Celsius. After 3 hours, I went ahead and placed my product in the -20 degrees Celsius freezer and will conduct Nanodrop and continue on with the protocol tomorrow.

9-28-12 100112 - Rishi - I would recommend doing cloning with this. When you have some waiting time - then re-do PCR squared and gel extract it for a second attempt at cloning. -- Dr. B My Nanodrop results are shown with a concentration of 134.2 ng/uL and absorbance ratio 260/280 = 1.90 and absorbance ratio 260/230 = 2.16. I may have to redo PCR squared because the results even after the nanodrop do not decipher the contaminants in my final product.

PCR squared was successful! I understand that the faded bands on the bottom may be problem when performing cloning. Thus, Gel Extraction will take place. My next step now is PCR Cleanup.

Lane 1: None Lane 2: 100 bp ladder Lane 3: PCR Product Lane 4: PCR Product Lane 5: PCR Product Lane 6: PCR Product Lane 7-10: None

9-27-12 PCR squared was conducted. The Gel Electrophoresis will take place tomorrow.

9-24-12 Virtual Drug Screening review session took place. I used pymol to interpret the results for each step from GOLD.

9-21-12 - Rishi - congrats. Good work.. -- Dr. B Success!! My PCR worked. I corrected the annealing temperature as well as the dilution factors for the forward and reverse primers. PCR^2 will take place next. More detail for today is stated in journal. Lane 1: None Lane 2: 100bp ladder Lane 3: None Lane 4: Primary PCR Lane 5: Secondary PCR Lane 6-10: None

9-19-12 Performing PCR __#|again__, I went back to the protocol using KOD Polymerase. I have prepared my tubes to conduct PCR as soon as the polymerase is added on Friday. Hopefully, my gel will show the correct bands.

9-14-12 Rishi - ok - maybe switch back to the KOD Hot Start? - Dr. B 091812 Primary and Secondary PCR Overlap Using tcGADPH Primers and Q5 Polymerase Lane 1: None Lane 2: 100bp ladder Lane 3: Primary PCR (nothing showed) Lane 4: Secondary PCR (nothing showed) Lane 5: None Lane 6-10: Max PCR (nothing showed as well)

Unfortunately, my Primary and Secondary PCRs did not show in the Gel Electrophoresis. Errors may have come from no amplification of the DNA used. Also, errors may have come from adding too many primers to the secondary PCR. I will conduct this protocol again and using caution in order to obtain accurate results. (More explained in journal.)

9-12-12 Primary PCR and Secondary PCR using KOD Polymerase was stopped. I realized that for the Secondary PCR, I used my template oligo mix instead of using the F primer and R primer from my target. As of right now, I am performing PCR using Q5 Polymerase.

9-7-12 Primary PCR and Secondary PCR were completed using KOD Polymerase. (There was one tube of KOD poly. remaining, so I decided to use it.) I will use the Q5 Polymerase if my gel does not turn out correctly.

9-5-12 Starting Oligonucleotide Mix Protocol. Pymol Refresher Protocol has been finished and is on Google Docs page. The main enzyme images are in my notebook with the appropriate captions.

8-2-12 Nanodrop: fthap, Trial 2 (Protein Purification) Measure 2 of the Nanodrop had a concentration of 0.08 mg/mL. With a wavelength of 280nm and an absorbance of 0.081, the yield for this particular trial was from the remainder of the centrifuge tube.

8-2-12 Nanodrop: fthap, Trial 1 (Protein Purification) Measure 1 of the Nanodrop had a concentration of 0.37 mg/mL. With a wavelength of 280nm and an absorbance of 0.366, the yield of fthap protein was significant for trial 1 and had the remainder in trial 2.

070212 - Rishi, put rough dates on your experiments here. Also, embed your images instead of the WORD documents. Any results from second attempt? -- Dr. B

6-29-12 PCR Overlap //Trypanosoma cruzi// (GAPDH) (2nd attempt)

(link to second attempt) Lane 1: 100bp ladder Lane 2: Primary PCR Lane 3: Secondary PCR, Option A Lane 4: Secondary PCR, Option B Lane 5: (Contamination occurred) Lane 6-10: Skipped

6-27-12 PCR Overlap //Trypanosoma cruzi// (GAPDH) (1st attempt) Lane 1: Skipped Lane 2: 100 bp ladder lane Lane 3-8: Michael and Max PCR Lane 9: Primary PCR (contamination may have occurred) Lane 10: Skipped

PCR-pGFP Lane 1: Skip Lane 2: 5 ul 100 bp ladder Lane 3: 0.016 ng total pGFP plasmid in 25 ul total test tube Lane 4: 0.16 ng total pGFP plasmid in 25 ul total test tube Lane 5: 1.6 ng total pGFP plasmid in 25 ul total test tube Lane 6: 0 ng total pGFP plasmid in 25 ul total test tube Lane 7: 0.016 ng total pGFP plasmid in 25 ul total test tube Lane 8: 0.16 ng total pGFP plasmid in 25 ul total test tube Lane 9: 1.6 ng total pGFP plasmid in 25 ul total test tube Lane 10: 0 ng total pGFP plasmid in 25 ul total test tube

6-20-12 PCR-pGBR22 062112 - Rishi - what plasmid is this PCR off of? - DR.B Lane 1: Skip Lane 2: 100bp DNA ladder Lane 3: 1 ul of 0.3 ng total pGBR22 plasmid Lane 4: 10 ul of 3.0 ng total pGBR22 plasmid Lane 5: 10 ul of 3.0 ng total pGBR22 plasmid in 25 ul total test tube Lane 6: 0 ul of 0.0 ng total pGBR22 plasmid in 25 ul total test tube

6-14-12 PCR-Restriction Enzyme Digest Lane 1: Skip Lane 2: Ladder Lane 3: PvuII cut Lane 4: PvuII & EcoRI cut Lane 5: EcoRI cut

Plate C: 1ng of Plasmid, 4000 colonies/ng

Plate B: 5ng of Plasmid, 2000 colonies/ng

Plate A: 25ng of Plasmid, 1600 colonies/ng


 * Nanodrop (pGBR22-purple protein) ** Trial 1


 * Nanodrop (pGBR22-purple protein) ** Trial 2

Lane 1: Skip Lane 2: 100bp DNA ladder Lane 3: 1 ul of 0.3 ng total pGBR22 plasmid in 25 ul total test tube (water, ThermoPol buffer, dNTP's, Taq, M13 Forward and Reverse Primers) Lane 4: 10 ul of 3.0 ng total pGBR22 plasmid in 25 ul total test tube (water, ThermoPol buffer, dNTP's, Taq, M13 Forward and Reverse Primers) Lane 5: 10 ul of 3.0 ng total pGBR22 plasmid in 25 ul total test tube (water, ThermoPol buffer, dNTP's, Taq, M13 Forward and Reverse Primers) Lane 6: 0 ul of 0.0 ng total pGBR22 plasmid in 25 ul total test tube (water, ThermoPol buffer, dNTP's, Taq, M13 Forward and Reverse Primers) PCR Overlap //Trypanosoma cruzi// (GAPDH)