ResearchPage+-+Ronnie

Ronnie's Research

 * Week 14: Virtual Screening 12/02/11**

The bestranking compounds from the three libraries was a popular hit within the LOPAC_3d library. The fitness scores are above 100.0 which means that ligands are most favorable for the inhibition assays.


 * Week 14: Enzyme Assays 12/01/11**

The data points form an increasing line. However, there are a couple of points that are not positively correlated to the rest of the data points.
 * Vary Substrate Phosphatase Enzyme Test**

The data increases in an exponential regression as the concentration increases. It is safe to continue to to the vary substrate enzyme test.
 * Phosphatase Enzyme Test**

These are terrible data. The absorbance of the decreases as the concentration increases on the interval [0,4.3] and then increases {4.3,9.8].
 * Phosphatase Enzyme Test**





MptpB is possibly in peaks 42-47. The other three peaks are probably run-offs from the filtration job ran before mine.
 * Week 13: FPLC Gel Filtration** **11/21/11**

It is kind of hard to view gel, but there is a distinct band for elution two.
 * Week 12: Protein Characterization** **11/18-11/19**

Began with a protein pH of 7.0 which was sufficient for the BIO-RAD chromophotography
 * Week 11: Protein Purification 11/09-11/10**

•Proceeding to protein purification and characterization to check the efficacy of the expression
 * Week 10: Protein Expression 11/02-11/04**

•If protein expression fails, IPTG concentration will be adjusted to 1mM and expressed for 6 hours at 30 oC to insure the activation of the lac promoter

Absorbance=0.318 at 1.5 hours Absorbance=0.869 at 2.0 hours
 * Spectrophotometry Measurement**


 * Transformation Plates**

Finally obtained a great concentration of protein with 227.6 ng/ul.
 * Week 10:** **Nanodrop Spectrophotometry 11/01/11**

In lane 7, I made a control secondary PCR composed of the leading and lagging Mtptb primer to validate whether the PCR worked. In lane 5, there were amplifications at around 1000 bp and below the DNA ladder. I suspect that the secondary PCR worked this time because I made new primers.
 * Week 10:** **Gel Electrophoresis 10/31/11**

Not good at at all. The secondary PCR is being amplified about 100 BP which is 10 times below what was expected.
 * Week 9:** **PCR Overlap Gel Electrophoresis 10/29/11**

Ronnie - keep trying - you will get it. But let's start shifting the majority of your effort over to protein expression. - Dr. B



This gel explains why my first two trials failed. The pNIC-Bsa4 were above the 1 KB DNA ladder. My next step is to wait 'til next week when Thou makes some new pNIC-Bsa4.
 * Week 9: pNIC-Bsa4 Gel Electrophoresis 10/28/11 **
 * Lane1:** 1 KB ladder (5ul)
 * Lane3:** pNIC-Bsa4 59.8 ng/ul (7ul)
 * Lane5:** pNIC-Bsa4 82.4 ng/ul (3ul)
 * Lane7:** 100 BP ladder (5ul)

I was successfully able to identify why my best ranking values were negative. The alpha helix of my pdb protein caused much torsional strain against the library of inhibitors. Now I am using pdb 2OZ5, which has an inhibitor already bound. Therefore, I can easily remove the inhibitor and dock others. I am still in the process of troubleshooting my runs and errors.
 * Week 9:** **Virtual Screening 10/28/11 **

Fatal error: licence request failed (for gold process creation): Flexlm raised the following error: Licensed number of users already reached Feature: gold License path: /apps/gold/goldsuite-5.0/ccdc_licence.dat:/apps/gold - /gold_v4.1/GOLD_Suite/ccdc_licence.dat:/apps/gold/gold_v4.0/GOLD_Suite - /ccdc_licence.dat FLEXlm error: -4,132 For further information, refer to the FLEXlm End User Manual, available at "www.macrovision.com". /apps/gold/goldsuite-5.0/ccdc_licence.dat

I made a new pNIC-Bsa4 accepting vector.There were blue dye in all of the lanes that were visible to the naked eye. I do not know why the DNA Ladder was the only thing that showed up.
 * Week 8: pNIC-Bsa4** Gel Electrophoresis **10/20/11**

Nothing showed up for the pNIC-Bsa4 vector in lane 9. I used the NEB enzymes so next time the Fermentus enzymes instead.
 * Week 8:** **pNIC-Bsa4** Gel Electrophoresis ** 10/18/11 **

I ran my GOLD run for the cb_306_3d and HF9library.
 * Week 8:** ** Protein Target Virtual Screen **

I ran my virtual screen Monday, but my run had a huge error file. Now my next step is to rerun the first run.
 * Week 7:** ** Protein Target Virtual Screen **


 * Week 7:** ** PCR Primer Overlap **

Same set-up as the fifth gel electrophoresis. It looks like there is a band in lanes 3 and 5 approximately 800-1000 bp. My best bet would probably be to PCR clean-up. It was brought to my attention on 10/19 that I was using the protein loading dye.
 * Sixth Gel Electrophoresis 10/16/11**

Secondary PCR (15ul) from primary PCR (1st Trial)
 * Nanodrop Spectrophotometry 10/16/11**

Tertiary PCR (15ul) from secondary PCR (1st Trial)
 * Nanodrop Spectrophotometry 10/16/11**

This time, I figured I would use the primary and secondary PCR from the first trial since my gel electrophoresis actually worked the first time. I made another secondary PCR from the primary PCR (1st trial) and then a tertiary PCR from the secondary PCR (1st trial). It was a failure so my next step is to repeat this step for verification.
 * Fifth Gel Electrophoresis 10/14/11**

This is very frustrated especially since I was like two weeks ahead of others, and now they have caught up with me. My gels are still not coming out the way I would like it to be. Hopefully, I will be able to finish the primer overlap Sunday.

Lane 7 is great, but it is the first trial that had a low concentration. I cant figure out why the 2nd and 3rd trials are not working.I am doing the same procedure.
 * Fourth Gel Electrophoresis 10/14/11**

Something went wrong with the gel. I'm guessing possibly cross-contamination.
 * Third Gel Electrophoresis 10/14/11**


 * Week 6:** ** PCR Primer Overlap Continued **

Evidently there was either an error in my protocol procedure or Joshua's MPTPb primary PCR was no good. This graph should look like the first gel electrophoresis with a distinct band at about 1000bp. The band shown at approximately 200bp is probably just primers. I also found my PCR samples from the first gel electrophoresis, but someone had left the PCR rack with my samples out at room temperature. My next step would be to start the PCR overlap all the way from scratch since my PCR samples might have denatured.
 * Second Gel Electrophoresis 10/05/11**

Ronnie - ok - good luck on getting it to work. -- Dr. B

The data was definitely too low of an absorbance. I was expecting the peak to have an absorbance close to 0.80nm.
 * Nanodrop Spectrophotometry 10/05/11**

I wanted to double check my results. Changes implemented: cleaned the Nanodrop spectrophotomer with ethanox, wiped with KimWipe, double blanked with water, pipetted 2ul of PCR-squared sample. Even though I did not receive the desired absorbance, the quantity did increase by a factor of 1.56nm. In the future, I would take extra caution with disinfecting my environment and equipment, and also reproofing my results or taking an average of trials experimented.
 * Nanodrop Spectrophotometry 10/05/11**

Unfortunately, none of the PCRs are shown and the 100bp DNA Ladder is barely displayed. I might just run another gel electrophoresis because I should have seen something. === ===
 * Third Gel Electrophoresis 10/06/11**


 * Week 6:** **PCR Primer Design for pNIC-Bsa4 Cloning**


 * Nucleotide Blast**



> [|emb|BX842572.1|] Mycobacterium tuberculosis H37Rv complete genome; segment 1/13 Length=341957

Features in this part of subject sequence: [|PHOSPHOTYROSINE PROTEIN PHOSPHATASE PTPB (PROTEIN-TYROSIN...]

Score = 503 bits (272), Expect = 9e-139 Identities = 646/829 (78%), Gaps = 16/829 (2%) Strand=Plus/Minus

Query 1 ATGGCGGTTCGTGAACTGCCGGGTGCGTGGAACTTTCGTGATGTTGCGGACACCGCGACC 60 ||||| || |||||||||||||| ||||||||||||||||| || || |||||||| ||| Sbjct 181985 ATGGCTGTCCGTGAACTGCCGGGCGCGTGGAACTTTCGTGACGTCGCCGACACCGCAACC 181926

Query 61 GCGCTGCGCCCTGGTCGTCTCTTCC-GTTCTTCTGAACTG-TCTCGCCTGGATGATGCCG 118 || |||| || || || || |||| || | | || ||| | ||||| || || |||| Sbjct 181925 GCATTGCGGCCGGGGCGGCTGTTCCGGTCCAGC-GAGCTGAGC-CGCCTCGACGACGCCG 181868

Query 119 GTCGTGCGACCCTCCGCCGTCTGGGTATCACCGATGTCGCGGACCTCCGCTCTTCTCGTG 178 | || || || || ||||| ||||| |||||||| || || ||||| || || || || | Sbjct 181867 GCCGGGCAACGCTGCGCCGGCTGGGGATCACCGACGTTGCCGACCTGCGGTCGTCCCGGG 181808

Query 179 AAGTTGCACGTCGTGGTCCAGGCCGCGTCCCGGACGGTATCGACGTTCACCTGCTGCCGT 238 | ||||| || || |||||||| || || |||||||| |||||||| ||||||||||||| Sbjct 181807 AGGTTGCCCGCCGCGGTCCAGGACGGGTTCCGGACGGCATCGACGTCCACCTGCTGCCGT 181748

Query 239 TCCCGGATCTGGCTGATGACGACGCGGATGA--CAGCGCTCCGCATGAAACCGCGTTCAA 296 |||| || || || ||||| ||||| || || ||||| ||||| |||||||| ||||| Sbjct 181747 TCCCCGACCTCGCCGATGATGACGCCGACGACTCAGCG--CCGCACGAAACCGCATTCAA 181690

Query 297 -ACGTCTGCTGACCAACGATGGCTCTAACGGTGAAAGCGGTGAAT-CTTCTCAGTCTATC 354 | | ||||| ||||| || || || ||||| || ||| |||| | | ||||| || Sbjct 181689 GA-GGCTGCTAACCAATGACGGGTCCAACGGCGAGTCCGGCGAATCCAGC-CAGTCGATA 181632

Query 355 AACGACGCAGCGACGCGTTACATGACCGACGAATACCGCCAATTTCCGACCCGCAACGGT 414 || ||||| || || || |||||||||||||| || |||||||| || || ||||| || Sbjct 181631 AATGACGCGGCCACCCGCTACATGACCGACGAGTATCGCCAATTCCCAACGCGCAATGGA 181572

Query 415 GCGCAACGTGCCCTGCATCGTGTAGTTACCCTGCTCGCGGCAGGCCGTCCAGTACTCACC 474 || || || || || |||||||| || || ||||| || || || || || || |||||| Sbjct 181571 GCACAGCGCGCGCTACATCGTGTCGTCACACTGCTTGCCGCCGGACGCCCGGTGCTCACC 181512

Query 475 CACTGTTTCGCGGGCAAAGACCGTACCGGTTTTGTGGTTGCGCTGGTTCTGGAAGCGGTT 534 ||||| |||||||| || || || ||||| || ||||| |||||||| || |||||||| Sbjct 181511 CACTGCTTCGCGGGTAAGGATCGCACCGGCTTCGTGGTCGCGCTGGTGCTTGAAGCGGTC 181452

Query 535 GGCCTCGATCGTGACGTTATCGTTGCAGACTACCTGCG-TTCTAATGACTCTGTTCCGCA 593 ||||| || || ||||| ||||| || ||||||||||| | || ||||| || || || Sbjct 181451 GGCCTGGACCGCGACGTCATCGTCGCCGACTACCTGCGCAGC-AACGACTCCGTGCCACA 181393

Query 594 GCTCCGTGCGCGTATCTCTGAAATGATCCAGCAGCGTTTCGACACCGAACTCGCGCCTGA 653 || || || || ||||| || ||||||||||||||||||||||||||||| || || || Sbjct 181392 ACTGCGGGCCCGGATCTCCGAGATGATCCAGCAGCGTTTCGACACCGAACTGGCACCCGA 181333

Query 654 AGTCGTTACCTTCACCAAAGCGCGTCTGAGCGACGGCGTTCTCGGTGTACGCGCTGAATA 713 || || || |||||||| || || ||| |||||| || || ||||| ||||| || || Sbjct 181332 GGTGGTGACGTTCACCAAGGCCCGGCTGTCCGACGGGGTCCTGGGTGTCCGCGCGGAGTA 181273

Query 714 CCTGGCGGCTGCGCGTCAGACCATCGACGAAACGTACGGCTCTCTGGGTGGCTACCTCCG 773 |||||| || || || |||||||| ||||| || ||||| || ||||| |||||||| || Sbjct 181272 CCTGGCCGCCGCACGCCAGACCATTGACGAGACCTACGGATCGCTGGGCGGCTACCTGCG 181213

Query 774 TGACGCGGGTATTT-CTCAGGCGACCGTTAATCGTATGCGTGGTGTGCT 821 ||||| ||||| | ||||| || || || || ||||| || ||||| Sbjct 181212 CGACGCCGGTATCAGC-CAGGCCACAGTCAACCGGATGCGCGGGGTGCT 181165

>lcl|15201 unnamed protein product Length=276
 * Protein Blast **

Score = 549 bits (1415), Expect = 2e-161, Method: Compositional matrix adjust. Identities = 276/276 (100%), Positives = 276/276 (100%), Gaps = 0/276 (0%)

Query 1 MAVRELPGAWNFRDVADTATALRPGRLFRSSELSRLDDAGRATLRRLGITDVADLRSSRE 60 MAVRELPGAWNFRDVADTATALRPGRLFRSSELSRLDDAGRATLRRLGITDVADLRSSRE Sbjct 1 MAVRELPGAWNFRDVADTATALRPGRLFRSSELSRLDDAGRATLRRLGITDVADLRSSRE 60

Query 61 VARRGPGRVPDGIDVHLLPFPDLADDDADDSAPHETAFKRLLTNDGSNGESGESSQSIND 120 VARRGPGRVPDGIDVHLLPFPDLADDDADDSAPHETAFKRLLTNDGSNGESGESSQSIND Sbjct 61 VARRGPGRVPDGIDVHLLPFPDLADDDADDSAPHETAFKRLLTNDGSNGESGESSQSIND 120

Query 121 AATRYMTDEYRQFPTRNGAQRALHRVVTLLAAGRPVLTHCFAGKDRTGFVVALVLEAVGL 180 AATRYMTDEYRQFPTRNGAQRALHRVVTLLAAGRPVLTHCFAGKDRTGFVVALVLEAVGL Sbjct 121 AATRYMTDEYRQFPTRNGAQRALHRVVTLLAAGRPVLTHCFAGKDRTGFVVALVLEAVGL 180

Query 181 DRDVIVADYLRSNDSVPQLRARISEMIQQRFDTELAPEVVTFTKARLSDGVLGVRAEYLA 240 DRDVIVADYLRSNDSVPQLRARISEMIQQRFDTELAPEVVTFTKARLSDGVLGVRAEYLA Sbjct 181 DRDVIVADYLRSNDSVPQLRARISEMIQQRFDTELAPEVVTFTKARLSDGVLGVRAEYLA 240

Query 241 AARQTIDETYGSLGGYLRDAGISQATVNRMRGVLLG 276 AARQTIDETYGSLGGYLRDAGISQATVNRMRGVLLG Sbjct 241 AARQTIDETYGSLGGYLRDAGISQATVNRMRGVLLG 276


 * Week 6:** **Virtual Screening**





Molecular Weight: 565.0582 [g/mol] Molecular Formula: C25H25ClN2O7S2 XLogP3-AA: 4.6 H-Bond Donor: 3 H-Bond Acceptor: 9


















 * Top 50 Bestranking**


 * Week 5:** ** PCR Primer Overlap -- Ronnie - good job. Don't need to post as much of the protocol - just focus more on your results like you show below in the image. Could label your gel lanes. -- Dr. B **


 * Gene of Interest: ** MptpB Rv0153c


 * Purpose: ** Synthesize a gene of interest from scratch


 * Objective: ** To clone your assigned gene using ov erlap extension PCR with the oligos designed using DNAworks

Continuing with expression vector option: You will go to ligation independent cloning (LIC) to insert the gene into the expression vector. Then you determine if it is correct by DNA sequencing.
 * Expression Vector Option B: ** pNIC-Bsa4


 * __ Oligo Mix __**
 * 1) Make a new microcentrifuge tube
 * 2) Add autoclaved Nanopure water to the tube. The amount of water will need to be equal to 100 - # of primers you have
 * For Example, if you have 20 primers for your gene, you would need 80 ul of water.
 * 1) Mix the contents of each primer in the deep well plate with a 200 ul pipette
 * 2) Remove 1 ul of each stock primer from the plate and add to the new ‘Primer Mix’ tube

**__ Primary PCR: __** 5 u l 10X rxn buffer 3 u l 25 mM MgSO4 5 u l 2 mM dNTPs 1 u l Template/Primers = 1 m M oligo mix 1 ul KOD hotstart Polymerase (1U/ m l) 35 u l sterile dH2O 50 u l Final Volume

Thermocylcer cycling conditions similar as primary PCR but the **__number of cycles is increased to 30__**.
 * __ Secondary PCR: __**

5 u l 10X rxn buffer 3 u l 25 mM MgSO4 5 u l 2 mM dNTPs 1 u l Template = primary PCR reaction (if more than one primary PCR reaction was set up, use 1ul from each primary PCR reaction) 1 u l 20 m M F primer (pNIC-Bsa4 custom primer 100 uM; 20 uM solution) 1 u l 20 m M R primer (pNIC-Bsa4 custom primer 100 uM ; 20 uM solution) 1 u l KOD hotstart Polymerase (1U/ m l) 33 u l sterile diH2O (adjust this volume if more than one primary PCR reaction was added to mix) 50 u l Final Volume

Some of the secondary PCR somehow have gotten into lane four which would explain the similar band in lane 3. === ===
 * First Gel Electrophoresis 09/30/11**




 * Week 4:** **Virtual Screening (Waiting on Second Run)**

Ronnie - show images instead of links to WORD docs - paste in your sequences for Primers here. . - Dr. B
 * Week 4: PCR Primer Design for Primer Overlap Assembly PCR**

__ Purpose __ : design an oligo set of forward and reverse primers for PCR synthesizing and amplifying the CDS of your gene of interest so that it can be inserted into a cloning (or expression) vector.

Go to the DNA Works website ([]) to upload your sequence. This site will take the amino acid sequence and back translate it into DNA. When this step is performed, the DNA is also ‘codon optimized’ to match the most preferred codons of the organism in which you will express your construct (most likely //E. coli// BL21(DE3) cells).

__ Job Name __ : put a descriptive job title e.g. **InitialsDateOrganismGeneName** (EPB061611Ecolidhfr) __ E-mail Address __ : in put your **email** (highly recommended) Skip __Mutant Run__: __ Codon Frequency Table __ : select ‘**E. coli class II’** __ Parameters __ : leave most as default except change ‘Oligo Length’ = **60 nt** Skip __Restriction Site Screen__: Skip __Custom Site Screen:__ Skip __Weights__: __ Sequences (s) (formats): __ select ‘**protein’** Then paste your amino acid sequence into the ‘Choice 2 – Enter sequence manually’ It is recommended that you ‘Filter’ your protein sequence first by going to the [|Sequence Manipulation Suite] and hitting ‘Filter Protein’ You need to be sure that a STOP amino acid is on the end (insert a ‘**X**’). Alternatively, we could add this in the amplification primers.

Hit ‘**Design Oligos**’

The DNA sequence # 1 is: 1 ATGGCGGTTCGTGAACTGCCGGGTGCGTGGAACTTTCGTGATGTTGCGGACACCGCGACC 61 GCGCTGCGCCCTGGTCGTCTCTTCCGTTCTTCTGAACTGTCTCGCCTGGATGATGCCGGT 121 CGTGCGACCCTCCGCCGTCTGGGTATCACCGATGTCGCGGACCTCCGCTCTTCTCGTGAA 181 GTTGCACGTCGTGGTCCAGGCCGCGTCCCGGACGGTATCGACGTTCACCTGCTGCCGTTC 241 CCGGATCTGGCTGATGACGACGCGGATGACAGCGCTCCGCATGAAACCGCGTTCAAACGT 301 CTGCTGACCAACGATGGCTCTAACGGTGAAAGCGGTGAATCTTCTCAGTCTATCAACGAC 361 GCAGCGACGCGTTACATGACCGACGAATACCGCCAATTTCCGACCCGCAACGGTGCGCAA 421 CGTGCCCTGCATCGTGTAGTTACCCTGCTCGCGGCAGGCCGTCCAGTACTCACCCACTGT 481 TTCGCGGGCAAAGACCGTACCGGTTTTGTGGTTGCGCTGGTTCTGGAAGCGGTTGGCCTC 541 GATCGTGACGTTATCGTTGCAGACTACCTGCGTTCTAATGACTCTGTTCCGCAGCTCCGT 601 GCGCGTATCTCTGAAATGATCCAGCAGCGTTTCGACACCGAACTCGCGCCTGAAGTCGTT 661 ACCTTCACCAAAGCGCGTCTGAGCGACGGCGTTCTCGGTGTACGCGCTGAATACCTGGCG 721 GCTGCGCGTCAGACCATCGACGAAACGTACGGCTCTCTGGGTGGCTACCTCCGTGACGCG 781 GGTATTTCTCAGGCGACCGTTAATCGTATGCGTGGTGTGCTCCTGGGTTAA




 * Week 4:** **Protocol of 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

Base pair 1400-1600 bp amplification was probably due to strong concentration of primers, and not diluting. This gel electrophoresis was my best so far since I improved my pipetting technique.
 * First Gel Electrophoresis 09/23/11**


 * Week 4:** **Virtual Screening (Started First Run)**


 * Week 3: Pymol Refresher**

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

**__ Background __** 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. **__ 2H2Q __**__ **3:08p.m. 09/15** __ 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.



**__ 3CL9 5:36p.m. __** This is the PDB identifier for the A chain (DHFR portion) with a known inhibitor (MTX). Make an image showing all of the components and then the polar contacts between the inhibitor and protein. Highlight the active site (around 5 angstroms from ligand) in a different color. $select active, MTX around 5 #code to select active site around object ‘MTX’

** Comparison to Human ** **__ 1U72 6:21 p.m. __** This is the PDB identifier for Human DHFR with MTX. Create selections for all of the components. Then, highlight the active sites (around 5 angstroms from ligand) in different colors. Then, bring the two proteins together by performing an alignment to show how closely the //T. cruzi// and the //H. sapiens// structures line up (compare 1U72 and 3CL9). __Record the RMS value__. How close are the binding modes of MTX to each of these two enzymes? Do you think the enzymes could be differentially targeted with a single drug? Are there any differences in the amino acid sequence in the active site (i.e. amino acids that come in contact with the ligand)? To determine the active site, click on your active site selection and then copy down the amino acids that are highlighted in the Sequence Viewer above. Show a pairwise comparison of the active site sequences in your report. Are there any similarities/differences between them?






 * Fig 5. ** Pairwise comparison of the active site sequences of 1U72 (yellow) and 3CL9 (green). One differnce in the amino acid sequence in the active site is serine in 1U72 and lysine in 3CL9.

**__ 3HBB 7:14 p.m. __** This is the PDB identifier for the complex with another known inhibitor (TMQ). Make a PyMol image showing all of the components and polar contacts between the protein and inhibitor. Display each chain distinctively. Highlight the active site (around 5 angstroms from ligand) in a different color. Is the binding mode of TMQ to //T. cruzi// DHFR-TS significantly different than that of MTX to human DHFR from 1U72? Address polar contacts and relevant amino acids in the active site.



Ronnie - those are some pretty gels! Good job documenting your work. Good luck on the next attempt. - Dr. B


 * Week 2: PCR Protocol**

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

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

I used M13 forward and reverse primers. Gel broke and lanes 3-6 did not show.
 * First Gel Electrophoresis 09/07/11**

Day 1 1) Digestive Reactions 2) Make digestion in centrifuge tube 3) Incubate for 1-2 hours 4) Heat block
 * Restrictive Enzyme Digest**
 * Eco RI
 * PvuII
 * EcoRI+PvuII

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

This time I used SP6 promotor and T7 promoter primers. The results were good, however there were probably poor methods of administration. I'm speculating that the samples did not settle into the wells correctly.
 * Second Gel Electrophoresis 09/09/11**

1) Place order for M13-For-20 (Order# 71971) online 2) Prepare template+M13-For primer+Nanopure 3) Deliver tube to DNA sequencing facility
 * Protocol Submitting DNA to DNA Sequencing Facility**




 * Week 1: Analyzing DNA Sequence of Plasmid**

Objective-Determine the DNA sequence of a plasmid

TATNGNATACTCAAGCTATGCATCCAACGCGTTGGGAGCTCTCCCATATGGTCGACCTGCAGGCGGCCGCACTAGTGATTTTGATTGATTGAAGG AGAAATATCATGAGTGTGATCGCTAAACAAATGACCTACAAGGTTTATATGTCAGGCACGGTCAATGGACACTACTTTGAGGTCGAAGGCGAT GGAAAAGGAAAGCCTTACGAGGGGGAGCAGACGGTAAAGCTCACTGTCACCAAGGGTGGACCTCTGCCATTTGCTTGGGATATTTTATCACCACTGTCTCAATACGG AAGCATACCATTCACCAAGTACCCTGAAGACATCCCTGATTATGTAAAGCAGTCATTCCCTGAGGGATATACATGGGAGAGGATCATGAACTTTGAAGATGGTGCAG TGTGTACTGTCAGCAATGATTCCAGCATCCAAGGCAACTGTTTCATCTACAATGTCAAAATCTCTGGTGTGAACTTTCCTCCCAATGGACCTGTTATGCAGAAGAAGC ACAGGGCTGGGAACCCAACACTGAGCGTCTCTTTGCACGAGATGGAATGCTGATAGGAAACAACTTTATGGCTCTGAAGTTGGAAGGAGGTGGTTACTATTTGTGTGA ATTCAAATCTACTTACAAGGCAAAGAAGCCTGTGAGGATGCCAGGGTATCACTATGTTGACCGCAAACTGGATGTAACCAGTCACAACAAGGATTACACATTTGTTGA GCAGTGTGAAATATCCATTGCACGCCACTCTTTGCTCGGTCATCACCATCACCATCACTAAAATCCCGCGGCCATGGCGGCCGGGAGCATGCGACGTCGGGCCCAAT TCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCT TTCGCCAGCTGGCGTAATAGCGAANAGGCCCGCACCGATCGCCCTTCCCACAGTTGCGCAGCCTGATGGCNATGGACGCNNCCTGNANCGNCGCATTAGCNCGGCN GGNNNGNGNNCNCGCAGCGNGACNCTACANTTNCNGCNNCNANC
 * __M13R DNA Sequence__**

**__Nucleotide Human Blast__** Human G+T (4 databases)



 * __Nucleotide Collection__**


 * __Purple Protein Coding Sequence Translation Results__**


 * __Slicing and Dicing__**

The gray regions are the areas that were cut off by the restriction enzymes. Both of the restriction enzymes have 100% activity in NEB buffer.


 * **__Eco RI__**




 * **__PvuII__**


 * **__PvuII+Eco RI__**