ResearchPage+-+TJ

TJ's Research

 * Week 14: Assay Tests**



The data points on the graph present an increasing trend; however there is a large standard deviation. There a a lot of data points within the 0.6 absorbence range which may show that there is some activity around that value.
 * Vary Substrate Phosphatase Enzyme Test **


 * Excel Chart with Values**


 * Week 13: Protein Purification/Characterization Images**








 * Week 12: Continuation With Protein**

Proceeded to protein purification and characterization. Sample 2 (Soluble Fraction) was lost so it was not included in the PAGE Gel.

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 * Week 11: Virtual**




 * Week 10: Protein Expression**



Day 3 completed Materials & Methods for cloning completed

Day 2 started and completed
 * Week 9: Protein Expression**


 * Week 8: Virtual Screening**

Virtual Screening on target started however waiting for run to start (error: need license)

The graph shows that I have a very high concentration
 * Week 7: PCR Overlap Nanodrop**

Primer Overlap started on **10/7.** Oligo mix created so PCR can be ran.
 * Week 6: Primer Overlap**


 * 10/14** Primary PCR and Secondary PCR ran in gel in order to check if the process was working. The next step is to perform PCR squared.



This image shows the oligo mix was made correctly. The primary PCR product looks like a smear and the secondary PCR has a distinct band that corresponds to the size of the gene. It is safe to move on to the PCR squared.


 * Primer Design:**
 * Oligonucleotides for Histidine Phosphatase (Francisella Tularensis)**

26 oligonucleotides need to be synthesized 1 ATGAAGAAAATCTTCGTTTCTTTCACGCTGCTGTTCTTCCTGATCCCTGTTGGTTACTC 59 2 CCGTGACGGGTGATCATAGACACGAAAATCAGTTTAGAAGAGTAACCAACAGGGATCAGG 60 3 CTATGATCACCCGTCACGGTGACCGTGCACCGTTCGCCAACATCGAAAACGCGAACTATT 60 4 GCCAATCGGGGTCAGTTCAGACAGTTCGGTACCCCAAGAATAGTTCGCGTTTTCGATGTT 60 5 GAACTGACCCCGATTGGCATGAACCAGGAATACAACCTGGGTCTCCAGCTGCGTAAACGT 60 6 GTCAACGTAATGTTCCGGGAGCAGACCGAATTTATCGATGTAACGTTTACGCAGCTGGAG 60 7 TCCCGGAACATTACGTTGACCAGTCTATCTACGTTCTGTCTTCTCACACCAACCGTACCG 60 8 CAGCAGGGTACAGACCCATCAGCAGAGACTGGGCGCTAACTACGGTACGGTTGGTGTGAG 60 9 TGGGTCTGTACCCTGCTGGCACCGGTCCGCTGATCGGTGACGGCGACCCAGCGATCAAAG 60 10 AGTCCGCAGACAGGGTCATAATCGGGATCGGCTGGAAACGATCTTTGATCGCTGGGTCGC 60 11 GACCCTGTCTGCGGACTCTCGTCTGATCCAGTTCCCGTACGAACAGTACCTCGCGGTTCT 60 12 GTTTTGTTTTGCCATTCCGGAGAATTGTAAACATACTTTTTGAGAACCGCGAGGTACTGT 60 13 TCCGGAATGGCAAAACAAAACCAAAGAAGCGGCTCCGAACTTTGCGAAATGGCAGCAGAT 60 14 CGGTGATAACGTCGTTCAGGCCAGAAATACGATTACCCAGGATCTGCTGCCATTTCGCAA 60 15 CCTGAACGACGTTATCACCGTTGGTGATGTCCTGATTGTTGCGCAGGCGCACGGTAAGCC 60 16 CGATGATTTGGTCCGCGTCTTCTTGAGAGAGACCTTTCGGCAGAGGCTTACCGTGCGCCT 60 17 ACGCGGACCAAATCATCGCGCTCACCGACTGGGGTCTGGCACAGCAATTTAAGTCTCAGA 60 18 GCGATTGGTGAGTTTACCACCCATGATGTAAGAAACTTTCTGAGACTTAAATTGCTGTGC 60 19 GTGGTAAACTCACCAATCGCATGATTGAAGACCTGAACAACGCGGTTAATGGTAAGTCTA 60 20 GAGTCAGGTCGTGACCAGAGTAGTAGGTCATTTTGTACTTAGACTTACCATTAACCGCGT 60 21 TCTGGTCACGACCTGACTCTGCTCGAAGTTATGGGCACCCTGGGTGTTCCGCTCGATACC 60 22 TTTGTACAGTTCCATTTCCAGGTTAGACGCGTAACCCGGTGCGGTATCGAGCGGAACACC 60 23 CCTGGAAATGGAACTGTACAAAGATGGTGATATCTACACGGTTAAACTGCGTTACAACGG 60 24 GAGTTGTTTTTGTCCATAATAGGGAGTTTGACGTATTTACCGTTGTAACGCAGTTTAACC 60 25 TCCCTATTATGGACAAAAACAACTCTTGCTCTCTGGACGCGCTGAATAAGTATATGCAAT 60 26 TTACTTCTGGAATTTTTCGTTAATAGATTGCATATACTTATTCAGCGCG 49


 * Week 5: Virtual Refresher**


 * Virtual Screening (Started 1st Run)**

Blades were full for a couple of days and when the first run was checked, there was an error. So refresher was restarted. Results coming soon!

PCR was ran again after failing the 1st time. Lane 6 is supposed to be control for PCR. May have added extra sample into loading well. There are also three bands which is unusual.
 * PCR**


 * Week 4:** **PCR for pNIC-Bsa4 cloning off of Cloning Vector**

Steps 1) Select primers: VDS15 For: CA2 Human, pNIC-Bsa4 (2.5 uM) VDS16 Rev: CA2 Human, pNIC-Bsa4 (2.5 uM) 2) Make master mix 3) Add master mix to each tube 4) Add DNA template to each tube (ID:4190, 383.1 ng/ul) 5) Add water 6) Add MgSO4 7) Keep on ice 8) Preheat PCR machine 9) Add Taq polymerase dilution (2ul+8ul Nanopure) 10) Run PCR machine 11) Make Agarose Gel

With respect to the image above, it's clear to see that the experiment failed. This may have happened because of contamination or incorrect preparation of dilutions (1:10; 1:100; 1:1000).

[[image:N3231_fig1_v1_000034.gif]]

 * Week 3: PyMol Results**

Examine three dimensional structure of a new enzyme
 * __Objective __**

DHFR-TS from //Trypanosoma cruzi// is a bi-functional enzyme complex that carries out the role of dihydrofolate reductase and thymidylate synthase. //T. cruzi// is the pathogen responsible for Chagas disease (also called American trypanosomiasis), which causes approximately 50,000 deaths annually. The disease is endemic in South and Central America. In the chronic form, Chagas disease causes severe damage to the heart and other organs. There is no satisfactory treatment for chronic Chagas disease and no vaccine is available. Potentially, this target could be used to inhibit growth of the parasite.
 * __Background __**

This is the PDB identifier for the complex with the natural substrates. Make a PyMol image showing all of the components separately (each component should be selected individually and given a name). Display each chain distinctively. Show polar contacts between the protein and any substrates or cofactors.
 * __9-15-11 2H2Q __**__ **3:00pm** __





It appears as if 1U72 and 3CL9 are similar when with respect to alignment scores. The similarities are that the active sites and the orientations of the MTX are extremely similar while some differences are that just a few amino acids are different in the active site. It’s also important to note that MTX is also the inhibitor for both of the proteins. Polar contacts help determine the binding affinity for different active sites. Essentially MTX has a stronger binding affinity for the active site of DHFR than TMQ to DHFR-TS.
 * Conclusion: **


 * Week 2: RE Digest**

Day 1 1) Digestive Reactions 2) Digestion in 1.7 ml centrifuge tube 3) Incubate at 37 degrees Celsius for 1-2 hours 4) Heat block for 20 min
 * Eco RI
 * PvuII
 * EcoRI+PvuII (double digestion in parallel)

Day 2 1) Make Agarose Gel 2) Run gel for about 40-45 min




 * Week 1: PCR Protocol**

Day 1 1)Make Template Dilutions 2) Make Master Mix 3) Add Master Mix to each tube 4) Add template to each tube 5) Place on ice 6)Preheat PCR machine 7) Add Taq polymerase 8) Run PCR machine

Day 2 1) Make Aragose Gel 2) Run Gel for about 40 min

T.J. - what is your template for this PCR below? Also, what is special about Sample D? How is Sample A,B,C different? - Dr. B

Aragose Gel: 9/7/11

Used 426.3 ng/micro liter of pGBR22 template. M13 forward and reverse primers (primer mix) were used. There is a clear distinction of where the band is. Shows up in the secondary lanes (3-5) which prove that the PCR worked. The best lane to chose from would be lanes 4 (sample C) and 5 (sample D) simply because of less fragmentation. The ladder on the gel also shows up very good as well.

Also show a clear band within lanes 3-5.