Tom+S.

Week 13+14 - Did enzyme and inhibition assay. Enzyme essay result:

Inhibition assay

Week 13 112812 - Concentrated the protein after FPLC to 1ml. - the concentration got higher. Guess it worked! - Gel image of our protein characterization Lane 1: n/a Lane 2: ladder Lane 3: sample 1 Lane 4: n/a Lane 5: sample 3 Lane 6: sample 4 Lane 7: n/a Lane 8: elution 1 Lane 9: elution 2 Lane 10: n/a

Week 12 112612 - Good. Dr B - Did Finished our protein expression/purification/characterizaiton. - Finished FPLC and concentrated protein is stored. Here is the NanoDrop result right after FPLC without concentration.

Week 11 Tom e- include some data or results here:--Dr. B 11/19/12 - Continue on Homology model. - Start on M&M cloning protocol

Week 10 - Protein expression protocol started. (FtHAP enzyme surrogate) - Homology model code 1auiA > [Template] |1auiA|2.1| HUMAN CALCINEURIN HETERODIMER Length = 378

[|[Display Alignment in DeepView]] Score = 336 bits (861), Expect = 2e-92,   Method: Composition-based stats. Identities = 165/372 (44%), Positives = 234/372 (62%), Gaps = 26/372 (6%)

Query: 17 PPPLWCEAKREALIDGKGKVNLEVMLVHFLRQGRLSKHDALNIIQNASSVLRTEPNVLRI 76 PP    AK   + D  GK  ++++  H +++GRL +  AL II   +S+LR E N+L I Sbjct: 11  PPSHRLTAKE--VFDNDGKPRVDILKAHLMKEGRLEESVALRIITEGASILRQEKNLLDI 68

Query: 77 ADPSVVVVGDVRGQFYDLAKIIFIGNMFSRAKT-YLFLGNYIDSCFFSTECILLLLAAKL 135 D V V GD+ GQF+DL K+  +G   S A T YLFLG+Y+D  +FS EC+L L A K+ Sbjct: 69  -DAPVTVCGDIHGQFFDLMKLFEVGG--SPANTRYLFLGDYVDRGYFSIECVLYLWALKI 125

Query: 136 THPSCVFLLRGNHECRFMSNIFDFRGECLKKYNDDVYEAIMTAFDCLPLAAVVNNQYFCV 195 +P +FLLRGNHECR ++  F F+ EC  KY++ VY+A M AFDCLPLAA++N Q+ CV Sbjct: 126 LYPKTLFLLRGNHECRHLTEYFTFKQECKIKYSERVYDACMDAFDCLPLAALMNQQFLCV 185

Query: 196 HGGLSPDVTSVDNIRLIYRFREPPSKGAMCDLLWSDPFWDVENPSSVCEGSRDDYYTPGN 255 HGGLSP++ ++D+IR + RF+EPP+ G MCD+LWSDP D  N Sbjct: 186 HGGLSPEINTLDDIRKLDRFKEPPAYGPMCDILWSDPLEDFGNEK--- 230

Query: 256 GPSYGTAPCFLDNEQRGCSYLFNYHSVKHFLLTNGLLCVVRSHEVQDDGYKLYRFNSVSN 315 T  F  N  RGCSY ++Y +V  FL  N LL ++R+HE QD GY++YR +  + Sbjct: 231 -TQEHFTHNTVRGCSYFYSYPAVCEFLQHNNLLSILRAHEAQDAGYRMYRKSQTTG 285

Query: 316 FPCMMSVFSAPNYCDNLHNKGAVLILEGKQIGIKQFCCSPHPYVLPKHLNAFEWSFSYLL 375 FP ++++FSAPNY D +NK AVL  E   + I+QF CSPHPY LP  ++ F WS  ++ Sbjct: 286 FPSLITIFSAPNYLDVYNNKAAVLKYENNVMNIRQFNCSPHPYWLPNFMDVFTWSLPFVG 345

Query: 376 DSVRDIFASIIS 387 + V ++ ++++ Sbjct: 346 EKVTEMLVNVLN 357 code

= SWISS-MODEL Workspace = Welcome tomjonggu@gmail.com * [| Documentation] Workunit: P000002 - Overview Print/Save this page as   Model Summary
 * ||
 * [|Repository]
 * [|Tools]
 * [|Modelling] || [|[ myWorkspace]] || Due to major network maintenance, interruptions of service are expected ||> [|[ Settings]] [|[ logout]] ||

|| || Only user specified template was used for modelling. || Model built :SINGLE CHAIN || Ligands in the model: none. ||  ||   ||
 * ||  || **Model information:** ||   ||
 * || Modelled residue range: || 9 to 388 ||
 * || Based on template: || [1auiA] (2.10 Å) ||
 * |||| **Remark:** No search for template was performed.
 * || Sequence Identity [%]: || 42.37 ||
 * || Evalue: || 0.00e-1 ||
 * || **Quality information:** ||  ||
 * || QMEAN Z-Score: -3.77 || [[image:http://swissmodel.expasy.org/workspace/img/qmean-zscore-background-s.png align="center"]] ||  ||   || **Quaternary structure information:** ||   ||
 * Template (1aui): HETERO DIMER
 * Template (1aui): HETERO DIMER
 * **Ligand information:** ||  ||
 * Ligands in the template: FE: 1, ZN: 1.
 * Ligands in the template: FE: 1, ZN: 1.

 Global Model Quality Estimation
 * **logs:**
 * display model:** as [|[pdb]] - as [|[DeepView project]] - in
 * download model:** as [|[pdb]] - as [|[Deepview project]] - as [|[text]] ||

|||||| **QMEAN4 global scores:** Z-Score: -3.77 Plot 1: [|[save png]] Plot 2: [|[save png]] ||= [|[save png]] ||= ||= [|[save png]] || [|[save jpg]] [|[save pdb]] || Energy profile: [|[save raw scores]] || **QMEAN4 global scores:** If you publish results from QMEAN, please cite the following paper: //Benkert P, Biasini M, Schwede T. (2011). "Toward the estimation of the absolute quality of individual protein structure models." Bioinformatics, 27(3):343-50.//
 * Local scores ||
 * QMEANscore4 [[image:http://swissmodel.expasy.org/workspace/img/help_b.png caption="?"]] || Estimated absolute model quality [[image:http://swissmodel.expasy.org/workspace/img/help_b.png caption="?"]] || Score components [[image:http://swissmodel.expasy.org/workspace/img/help_b.png caption="?"]] || Coloring by residue error [[image:http://swissmodel.expasy.org/workspace/img/help_b.png caption="?"]] || Residue error plot [[image:http://swissmodel.expasy.org/workspace/img/help_b.png caption="?"]] ||
 * = 0.54 ||= [[image:http://swissmodel.expasy.org/workspace/index.php?func=getFile&file=getStructAFile&userid=tomjonggu@gmail.com&prjid=P000002&token=f0052b77db8ae4f80053b1c43f4680c1&key=0b94deb0f9b55d5d6786a58b86151055&model=Batch.1&name=QMEAN_plots/Batch.1.short.pdb_plot.png_thumb.png&show=png]]
 * || Coloring (all chains):
 * The QMEAN4 score is a composite score consisting of a linear combination of 4 statistical potential terms (estimated model reliability between 0-1). The pseudo-energies of the contributing terms are given below together with their Z-scores with respect to scores obtained for high-resolution experimental structures of similar size solved by X-ray crystallography: ||
 * Scoring function term ||= Raw score ||= Z-score ||
 * C_beta interaction energy ||= -68.40 ||= -1.48 ||
 * All-atom pairwise energy ||= -5657.95 ||= -2.05 ||
 * Solvation energy ||= -7.57 ||= -2.81 ||
 * Torsion angle energy ||= -51.94 ||= -2.40 ||
 * QMEAN4 score ||= 0.539 ||= -3.77 ||

 Local Model Quality Estimation: Anolea / QMEAN / Gromos:

Download raw data: [|[Anolea Data]] [|[QMEAN Data]]
 * anolea: on off QMEAN: on off gromos: on off ||

 Alignment

 Modelling Log

 Template Selection Log

 Quaternary Structure Modeling Log

 Ligand Modeling Log References If you publish results using SWISS-MODEL, please cite the following papers: - Arnold K., Bordoli L., Kopp J., and Schwede T. (2006). The SWISS-MODEL Workspace: A web-based environment for protein structure homology modelling. Bioinformatics, 22,195-201. - Schwede T, Kopp J, Guex N, and Peitsch MC (2003) SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Research 31: 3381-3385. - Guex, N. and Peitsch, M. C. (1997) SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modelling. Electrophoresis 18: 2714-2723.


 * ||  Swiss Institute of Bioinformatics | [|About SWISS-MODEL] | [|Help] | [|Terms of use] | News [|Back to the Top] ||   ||
 * ||  Swiss Institute of Bioinformatics | [|About SWISS-MODEL] | [|Help] | [|Terms of use] | News [|Back to the Top] ||   ||

Week 9 102912 - Re-checked my gel for primary PCR with both 56 and 57 degree celcius Lane 1: skipped Lane 2: 100bp buffer Lane 3-7: N/A Lane 8: 57 degree celcius Primary PCR Lane 9: 56 degree celcius Primary PCR

Week 8 102512- Finished my research report. 102612- Ran the Gel of my 2nd attempt of PCR for my target protein \ Lane 1: skipped Lane 2: skipped Lane 3: 100 bp ladder Lane 4: N/A Lane 5: primary PCR Lane 6: Secondary PCR
 * this time i lowered all the temperatures of all cycles. Messed up result... will try again with only 1 and 2 degree celcius down on annealing temperature.

102712- Ran my 3rd attempt of PCR primary with 57 and 56 degree celsius for annealing temperature. Apparently... the Gel did not contain any etBr... gonna run it again.

Week 7 102112 - Tom - ok - but show your gel images. -- Dr. B 101712- Did my Virtual Refresher. I started this on Monday and just ran the 2nd job. Hope to get a result by tomorrow to analyze. There are the top 10 ligands 101712- Also, i redid my primary PCR. I lowered the melting temperature by 1 degree celcius just to prevent any denaturation. I will run the gel for this and see if i get any result and will either keep going on or find out what's wrong with this.

Week 6 101612 - tom, ok try to figure out what is going on here and see if you can get Primary and Secondary to work. -- Dr. B I think we should sit down and double check the groups primer design to make sure there aren't any errors there.

100812- Started my primary PCR for my target. Sajan already made a oligo-mix for our protein (phosphatase 2B) so i used the sample. 101012- did my secondary PCR for the sample, waiting for the result 101212- Finished my 2nd PCR and have a gel image. The gel had Aldo and Brandon's sample also that im not sure if my PCR worked.

Week 5 100912 - Tom show some wet lab results too. DR. B -Did my PyMol refresher. PyMol Refresher Images. Figure 1. Image of 2H2Q molecule. Substrate is in red, hydrophobic with pink, ionic with blue, and polar residues w ith blue. Figure 2. Image of 3CL9 molecule. Inhibitor (MTX) is in pink, substrate with cyan, and active site with orange. Figure 3. 1U72 molecule image. Cyan is 3CL9 and green is 1U72. Orange is active site of 1U72 and blue is active site of 3CL9. The RMS value was 1.126. The enzymes could be differentially targeted with a single drug since the binding sites for both MTX in the molecules are similar. The active site was mostly overlapping except that 1u72 had asp but 3cl9 did not have them in the active site. Figure 4. BLAST result of 3CL9 and 1U72 Figure 5. Image of 3HBB molecule. The inhibitor (TMQ) is in pink, polar contacts with black, active site with orange. The binding mode of TMQ to T. cruzi DHFR-TS is not significantly different from that of MTX to human DHFR from 1u72.

Week 4 093012- Tom, good - label this gel a little better so that I know what the bands are. -- Dr. B Had 3 exams this week, so my lab effort was a little bit less devoted, forgive me coach. - Did 2nd PCR today (9.26/12) and left it at the lab for running. Will check the gel tomorrow for confirmation. failed PCR 2.....

- Did PCR primer design for pNIC - Bsa4 cloning The DNA sequence # 1 is:

1 ATGCTGCCAATCCTGAAAGCGGGTGAAGACGAGGGTGGTCCGCGTATCCCGCCACCTCTG

61 TGGTGCGAAGCGAAACGTGAAGCGCTGATCGACGGTAAAGGTAAAGTTAACCTGGAAGTC

121 ATGCTGGTTCACTTCCTCCGCCAGGGTCGTCTGTCTAAACACGACGCGCTGAACATCATC

181 CAAAATGCATCTTCCGTACTGCGTACCGAACCGAACGTCCTGCGTATCGCCGATCCGTCT

241 GTTGTTGTCGTTGGTGACGTACGCGGCCAATTCTATGACCTGGCGAAAATCATCTTCATC

301 GGTAATATGTTCTCTCGCGCCAAAACCTACCTGTTCCTGGGTAACTACATCGACTCTTGC

361 TTCTTCTCTACCGAGTGCATCCTCCTCCTGCTGGCGGCGAAGCTGACCCATCCTTCTTGC

421 GTATTCCTGCTCCGTGGCAACCACGAATGCCGTTTCATGAGCAACATTTTTGACTTTCGC

481 GGTGAATGCCTGAAGAAATACAACGACGACGTTTACGAGGCGATCATGACCGCGTTCGAC

541 TGCCTGCCGCTGGCCGCTGTCGTTAACAACCAATACTTCTGCGTTCACGGTGGCCTGTCC

601 CCGGACGTTACGTCTGTGGACAACATTCGTCTGATCTACCGTTTCCGCGAACCGCCTTCT

661 AAGGGTGCGATGTGTGATCTGCTGTGGAGCGACCCGTTCTGGGACGTTGAAAACCCGTCT

721 TCTGTTTGTGAAGGCTCTCGTGACGACTACTACACCCCAGGCAATGGTCCGTCTTACGGT

781 ACGGCGCCATGCTTCCTGGATAATGAACAACGCGGCTGTAGCTATCTGTTCAACTACCAC

841 TCCGTCAAACACTTTCTGCTCACTAACGGTCTGCTCTGTGTTGTTCGTTCTCACGAAGTT

901 CAGGACGACGGTTACAAACTGTATCGTTTTAACTCCGTGTCTAACTTTCCGTGCATGATG

961 TCTGTTTTCTCTGCGCCTAACTACTGCGACAACCTGCATAACAAAGGCGCGGTTCTGATC

1021 CTGGAGGGTAAACAGATCGGTATCAAACAGTTCTGCTGCTCCCCGCACCCGTACGTTCTG

1081 CCGAAACACCTCAACGCCTTCGAATGGTCTTTCAGCTACCTCCTGGACTCTGTTCGCGAC

1141 ATCTTCGCGTCTATTATCTCTTGCGAAGGTGCGTAA

--

Filtered: ATGCTGCCAATCCTGAAAGCGGGTGAAGACGAGGGTGGTCCGCGTATCCCGCCACCTCTG TGGTGCGAAGCGAAACGTGAAGCGCTGATCGACGGTAAAGGTAAAGTTAACCTGGAAGTC ATGCTGGTTCACTTCCTCCGCCAGGGTCGTCTGTCTAAACACGACGCGCTGAACATCATC CAAAATGCATCTTCCGTACTGCGTACCGAACCGAACGTCCTGCGTATCGCCGATCCGTCT GTTGTTGTCGTTGGTGACGTACGCGGCCAATTCTATGACCTGGCGAAAATCATCTTCATC GGTAATATGTTCTCTCGCGCCAAAACCTACCTGTTCCTGGGTAACTACATCGACTCTTGC TTCTTCTCTACCGAGTGCATCCTCCTCCTGCTGGCGGCGAAGCTGACCCATCCTTCTTGC GTATTCCTGCTCCGTGGCAACCACGAATGCCGTTTCATGAGCAACATTTTTGACTTTCGC GGTGAATGCCTGAAGAAATACAACGACGACGTTTACGAGGCGATCATGACCGCGTTCGAC TGCCTGCCGCTGGCCGCTGTCGTTAACAACCAATACTTCTGCGTTCACGGTGGCCTGTCC CCGGACGTTACGTCTGTGGACAACATTCGTCTGATCTACCGTTTCCGCGAACCGCCTTCT AAGGGTGCGATGTGTGATCTGCTGTGGAGCGACCCGTTCTGGGACGTTGAAAACCCGTCT TCTGTTTGTGAAGGCTCTCGTGACGACTACTACACCCCAGGCAATGGTCCGTCTTACGGT ACGGCGCCATGCTTCCTGGATAATGAACAACGCGGCTGTAGCTATCTGTTCAACTACCAC TCCGTCAAACACTTTCTGCTCACTAACGGTCTGCTCTGTGTTGTTCGTTCTCACGAAGTT CAGGACGACGGTTACAAACTGTATCGTTTTAACTCCGTGTCTAACTTTCCGTGCATGATG TCTGTTTTCTCTGCGCCTAACTACTGCGACAACCTGCATAACAAAGGCGCGGTTCTGATC CTGGAGGGTAAACAGATCGGTATCAAACAGTTCTGCTGCTCCCCGCACCCGTACGTTCTG CCGAAACACCTCAACGCCTTCGAATGGTCTTTCAGCTACCTCCTGGACTCTGTTCGCGAC ATCTTCGCGTCTATTATCTCTTGCGAAGGTGCGTAA

Forward sequence: TACTTCCAATCCATG CTGCCAATCCTGAAAGCG Reverse sequence: TATCCACCTTTACTG TTACGCACCTTCGCAAGA

Forward primer: TACTTCCAATCCATG CTGCCAATCCTGAAAGCG 33 bp, GC content: 48.5% Mg2+ temperature 0mM: 65 °C, 1.5mM: 72°C, 2mM: 72.5°C, 4mM: 73.4°C, 6mM: 73.9°C

Reverse primer: TATCCACCTTTACTG.TTACGCACCTTCGCAAGA 33 bp, GC content: 45.5% Mg2+ temperature 0mM: 63.8°C, 1.5mM: 71°C, 2mM: 71.5°C, 4mM: 72.5°C, 6mM: 73°C

Inserted version:

__ ATG ____ CTGCCAATCCTGAAAGCG __ GGTGAAGACGAGGGTGGTCCGCGTATCCCGCCACCTCTG TGGTGCGAAGCGAAACGTGAAGCGCTGATCGACGGTAAAGGTAAAGTTAACCTGGAAGTC ATGCTGGTTCACTTCCTCCGCCAGGGTCGTCTGTCTAAACACGACGCGCTGAACATCATC CAAAATGCATCTTCCGTACTGCGTACCGAACCGAACGTCCTGCGTATCGCCGATCCGTCT GTTGTTGTCGTTGGTGACGTACGCGGCCAATTCTATGACCTGGCGAAAATCATCTTCATC GGTAATATGTTCTCTCGCGCCAAAACCTACCTGTTCCTGGGTAACTACATCGACTCTTGC TTCTTCTCTACCGAGTGCATCCTCCTCCTGCTGGCGGCGAAGCTGACCCATCCTTCTTGC GTATTCCTGCTCCGTGGCAACCACGAATGCCGTTTCATGAGCAACATTTTTGACTTTCGC GGTGAATGCCTGAAGAAATACAACGACGACGTTTACGAGGCGATCATGACCGCGTTCGAC TGCCTGCCGCTGGCCGCTGTCGTTAACAACCAATACTTCTGCGTTCACGGTGGCCTGTCC CCGGACGTTACGTCTGTGGACAACATTCGTCTGATCTACCGTTTCCGCGAACCGCCTTCT AAGGGTGCGATGTGTGATCTGCTGTGGAGCGACCCGTTCTGGGACGTTGAAAACCCGTCT TCTGTTTGTGAAGGCTCTCGTGACGACTACTACACCCCAGGCAATGGTCCGTCTTACGGT ACGGCGCCATGCTTCCTGGATAATGAACAACGCGGCTGTAGCTATCTGTTCAACTACCAC TCCGTCAAACACTTTCTGCTCACTAACGGTCTGCTCTGTGTTGTTCGTTCTCACGAAGTT CAGGACGACGGTTACAAACTGTATCGTTTTAACTCCGTGTCTAACTTTCCGTGCATGATG TCTGTTTTCTCTGCGCCTAACTACTGCGACAACCTGCATAACAAAGGCGCGGTTCTGATC CTGGAGGGTAAACAGATCGGTATCAAACAGTTCTGCTGCTCCCCGCACCCGTACGTTCTG CCGAAACACCTCAACGCCTTCGAATGGTCTTTCAGCTACCTCCTGGACTCTGTTCGCGAC ATCTTCGCGTCTATT __ TCTTGCGAAGGTGCGTAACAGTAAAGGTGGATA __

pNIC Bsa4 FASTA TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGAGAACCTGTACTTCCAA T __ ATG ____ CTGCCAATCCTGAAAGCG __ GGTGAAGACGAGGGTGGTCCGCGTATCCCGCCACCTCTGTGGTGCGAAGCGAAACGTGAAGCGCTGATCGACGGTAAAGGTAAAGTTAACCTGGAAGTCATGCTGGTTCACTTCCTCCGCCAGGGTCGTCTGTCTAAACACGACGCGCTGAACATCATCCAAAATGCATCTTCCGTACTGCGTACCGAACCGAACGTCCTGCGTATCGCCGATCCGTCT GTTGTTGTCGTTGGTGACGTACGCGGCCAATTCTATGACCTGGCGAAAATCATCTTCATC GGTAATATGTTCTCTCGCGCCAAAACCTACCTGTTCCTGGGTAACTACATCGACTCTTGC TTCTTCTCTACCGAGTGCATCCTCCTCCTGCTGGCGGCGAAGCTGACCCATCCTTCTTGC GTATTCCTGCTCCGTGGCAACCACGAATGCCGTTTCATGAGCAACATTTTTGACTTTCGC GGTGAATGCCTGAAGAAATACAACGACGACGTTTACGAGGCGATCATGACCGCGTTCGAC TGCCTGCCGCTGGCCGCTGTCGTTAACAACCAATACTTCTGCGTTCACGGTGGCCTGTCC CCGGACGTTACGTCTGTGGACAACATTCGTCTGATCTACCGTTTCCGCGAACCGCCTTCT AAGGGTGCGATGTGTGATCTGCTGTGGAGCGACCCGTTCTGGGACGTTGAAAACCCGTCT TCTGTTTGTGAAGGCTCTCGTGACGACTACTACACCCCAGGCAATGGTCCGTCTTACGGT ACGGCGCCATGCTTCCTGGATAATGAACAACGCGGCTGTAGCTATCTGTTCAACTACCAC TCCGTCAAACACTTTCTGCTCACTAACGGTCTGCTCTGTGTTGTTCGTTCTCACGAAGTT CAGGACGACGGTTACAAACTGTATCGTTTTAACTCCGTGTCTAACTTTCCGTGCATGATG TCTGTTTTCTCTGCGCCTAACTACTGCGACAACCTGCATAACAAAGGCGCGGTTCTGATC CTGGAGGGTAAACAGATCGGTATCAAACAGTTCTGCTGCTCCCCGCACCCGTACGTTCTG CCGAAACACCTCAACGCCTTCGAATGGTCTTTCAGCTACCTCCTGGACTCTGTTCGCGAC ATCTTCGCGTCTATT __ TCTTGCGAAGGTGCGTAAC __ AGTAAAGGTGGATACGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAAT

Week 3 Tom - ok not sure your amplification on PCR worked. Try again and check that your cycling protocol is correct. Good jobincluding the ladder image on right. Try to crop the main image though. Nice job on Midiprep. -- Dr. B

My 1st PCR was thrown away by someone, so I will have to re-do this lab on friday.

Re-did my 1st PCR. Not sure if that is the right one though..

Midiprep was done today. The result Nanodrop image is provided below.

The sample was initially combined into one tube, so it might have been too concentrated.

Week 1 & 2

Other labs were done this week. Primer dilution, DNA sequence analysis (which is uploaded in the google docs).

somehow, my gel image can't be converted to jpeg... i dont know how to do that

Restriction Digest of pGBR22

Lane 1; Skip

Lane 2; 1 kb NEB DNA Ladder

Lane 3; Uncut pGBR22 plasmid

Lane 4; EcoRI-Hf

Lane 5; PvuII

Lane 6; EcoRI-Hf/PvuII

Personal target page:

Someone deleted my original target page so i had to generate another target page. It was about T. Brucei Here is the basic information about the target.


 * Disease: ** Trypansoma brucei


 * Enzyme: ** [|Protein Phosphatase 2B]


 * Divya - you need your gene ID info and protein ID info here. -- Dr. B **


 * Essentiality: ** Essential, used for [|cytokinesis]


 * Not complex **


 * EC#: ** 3.1.3.16

**Drugability:** 0.8 (yes)


 * Assay: **

[]

[]


 * crystal structure: **


 * [[image:vdsstream/3ll8_bio_r_500.jpg caption="3ll8_bio_r_500.jpg"]] ||
 * Fig. 1. ribbon structure of Protein phosphatase 2B also known as Calceneurin ||

[|**Amino Acid sequence:**]

length: 391

MLPILKAGEDEGGPRIPPPLWCEAKREALIDGKGKVNLEVMLVHFLRQGRLSKHDALNIIQNASSVLRTE PNVLRIADPSVVVVGDVRGQFYDLAKIIFIGNMFSRAKTYLFLGNYIDSCFFSTECILLLLAAKLTHPSC VFLLRGNHECRFMSNIFDFRGECLKKYNDDVYEAIMTAFDCLPLAAVVNNQYFCVHGGLSPDVTSVDNIR LIYRFREPPSKGAMCDLLWSDPFWDVENPSSVCEGSRDDYYTPGNGPSYGTAPCFLDNEQRGCSYLFNYH SVKHFLLTNGLLCVVRSHEVQDDGYKLYRFNSVSNFPCMMSVFSAPNYCDNLHNKGAVLILEGKQIGIKQ FCCSPHPYVLPKHLNAFEWSFSYLLDSVRDIFASIISCEGA