ResearchPage+-+Mohammed

Mohammed's Research

 * WEEK 13**






 * WEEK 12**



Serine Threonine Phosphotase 2C from http://web.expasy.org/cgi-bin/protparam/protparam


 * Number of amino acids:** 324


 * Molecular weight:** 35931.7


 * Theoretical pI:** 5.21






 * WEEK 11**

This week I finished my PCR squared and completed my PCR cleanup



And I finished up my protein expression. The Pellet Weight was 2.07g for sample 1 and 2.54 for sample 2.



It took an hour and thirty minutes to get to get to 0.1 absorption Sample 1 Sample 2 3:00 0.163 0.108 0.193 0.199 0.29 0.28



I forgot to take a picture of the pellet but I completed the procedure and the supernatant is in the 4 degree fridge. Also I had two samples but ended up adding too much lysate to sample one so I did my protein purification with sample 2.

WEEK 10

I was not in lab at all this week, well maybe for like 2 to 3 hours, but i will make the hours up for next week. Had three tests and I got pretty sick so sorry about that. I was supposed to do PCR squared but I will be finishing that next week. I did my slides for my presentation and talked to Dr.B about them. Finally fixed my virtual screening. I started on my Protein Expression but I left too long of a gap so I need to redo it for next week.

So for next week I will finish Virtual Screening of HF9 Library and most probably one other library now that I can do it myself. Present Finish all of the PCR Finish Protein Expression Will definitely start cloning but don't know if I will be able to finish.

__**WEEK 9**__ Mohammed - I think this should be Week 9 - spit up your Week 8 and 9 results. Good job on the cloning! - Dr. B

Lane 4: 100bp ladder Lane 5: 2nd PCR Lane 6: 1st PCR Lane 7: 1000kb ladder Lane 8: 1st PCR



Lane 1: 100 bp ladder Lane 2: 1000 kb ladder Lane 3: Primary PCR Lane 4: Secondary PCR



Lane 1: 100bp ladder Lane 2: 1kb ladder Lane 3: 1st PCR Lane 4: 2nd PCR

__**WEEK 8**__


 * Also ran the first GOLD job. Here is the gold.conf file for the first run.**

GOLD CONFIGURATION FILE

AUTOMATIC SETTINGS autoscale = 0.1

POPULATION popsiz = auto select_pressure = auto n_islands = auto maxops = auto niche_siz = auto

GENETIC OPERATORS pt_crosswt = auto allele_mutatewt = auto migratewt = auto

FLOOD FILL radius = 10 origin = 30.877 16.634 59.271 do_cavity = 1 floodfill_atom_no = 2454 cavity_file = ID_protein.mol2 floodfill_center = atom

DATA FILES ligand_data_file /home/chem204/DatabasesVDS/HF9PlatesPlates5_9.sdf 10 param_file = DEFAULT set_ligand_atom_types = 1 set_protein_atom_types = 0 directory =. tordist_file = DEFAULT make_subdirs = 0 save_lone_pairs = 1 fit_points_file = fit_pts.mol2 read_fitpts = 0 bestranking_list_filename = BestPP2CvsHF9Run1.1st

FLAGS internal_ligand_h_bonds = 0 flip_free_corners = 0 match_ring_templates = 0 flip_amide_bonds = 0 flip_planar_n = 1 flip_ring_NRR flip_ring_NHR flip_pyramidal_n = 0 rotate_carboxylic_oh = flip use_tordist = 1 postprocess_bonds = 1 rotatable_bond_override_file = DEFAULT solvate_all = 1

TERMINATION early_termination = 1 n_top_solutions = 3 rms_tolerance = 1.5

CONSTRAINTS force_constraints = 0

COVALENT BONDING covalent = 0

SAVE OPTIONS save_score_in_file = 1 save_protein_torsions = 1 concatenated_output = PP2CvsHF9Run1.sdf clean_up_option delete_all_solutions clean_up_option save_top_n_solutions 1 clean_up_option save_best_ligands 10 clean_up_option delete_redundant_log_files clean_up_option delete_all_initialised_ligands clean_up_option delete_empty_directories clean_up_option delete_rank_file clean_up_option delete_all_log_files output_file_format = MACCS

FITNESS FUNCTION SETTINGS initial_virtual_pt_match_max = 3 relative_ligand_energy = 1 start_vdw_linear_cutoff = 6 score_param_file = DEFAULT

PROTEIN DATA protein_datafile = /home/chem204/2011/ma32259/Serine Threonine Phosphotase 2C/ID_protein.mol2

Here is the gold.conf file for the second fun
 * Also ran the second GOLD Job**

GOLD CONFIGURATION FILE

AUTOMATIC SETTINGS autoscale = 1

POPULATION popsiz = auto select_pressure = auto n_islands = auto maxops = auto niche_siz = auto

GENETIC OPERATORS pt_crosswt = auto allele_mutatewt = auto migratewt = auto

FLOOD FILL radius = 10 origin = 30.877 16.634 59.271 do_cavity = 1 floodfill_atom_no = 2454 cavity_file = ID_protein.mol2 floodfill_center = atom

DATA FILES ligand_data_file /home/chem204/2011/ma32259/Serine Threonine Phosphotase 2C/SDFCOMBINED 10 param_file = DEFAULT set_ligand_atom_types = 1 set_protein_atom_types = 0 directory =. tordist_file = DEFAULT make_subdirs = 0 save_lone_pairs = 1 fit_points_file = fit_pts.mol2 read_fitpts = 0 bestranking_list_filename = BestPP2CvsHF9Run1.1st

FLAGS internal_ligand_h_bonds = 0 flip_free_corners = 0 match_ring_templates = 0 flip_amide_bonds = 0 flip_planar_n = 1 flip_ring_NRR flip_ring_NHR flip_pyramidal_n = 0 rotate_carboxylic_oh = flip use_tordist = 1 postprocess_bonds = 1 rotatable_bond_override_file = DEFAULT solvate_all = 1

TERMINATION early_termination = 1 n_top_solutions = 3 rms_tolerance = 1.5

CONSTRAINTS force_constraints = 0

COVALENT BONDING covalent = 0

SAVE OPTIONS save_score_in_file = 1 save_protein_torsions = 1 concatenated_output = PP2CvsHF9Run2.sdf clean_up_option delete_all_solutions clean_up_option save_top_n_solutions 1 clean_up_option save_best_ligands 1 clean_up_option delete_redundant_log_files clean_up_option delete_all_initialised_ligands clean_up_option delete_empty_directories clean_up_option delete_rank_file clean_up_option delete_all_log_files output_file_format = MACCS

FITNESS FUNCTION SETTINGS initial_virtual_pt_match_max = 3 relative_ligand_energy = 1 start_vdw_linear_cutoff = 6 score_param_file = DEFAULT

PROTEIN DATA protein_datafile = /home/chem204/2011/ma32259/Serine Threonine Phosphotase 2C/ID_protein.mol2

Best Ranking for 1st GOLD RUN
 * File containing a listing of the fitness of the top-ranked ||  ||   ||   ||   ||   ||
 * individual for each ligand docked in GOLD. ||  ||   ||   ||   ||   ||   ||
 * Format is: ||  ||   ||   ||   ||   ||   ||   ||   ||
 * Fitness || S(hb_ext) || S(vdw_ext) || S(hb_int) || S(int) || intcor || time || File name |||| Ligand name ||
 * 61.69 || 0 || 45.9 || 0 || -1.42 || 0.64 || 11.335 || 'output_1_200/gold_soln_HF9PlatesPlates5_9_m117_1.sdf' |||| 'JFD00064SC' ||
 * 59.65 || 0 || 44.07 || 0 || -0.94 || 12.33 || 57.45 || 'output_1_200/gold_soln_HF9PlatesPlates5_9_m170_5.sdf' |||| 'RJF00101SC' ||
 * 57.48 || 6 || 38.94 || 0 || -2.07 || -2.5 || 26.999 || 'output_201_400/gold_soln_HF9PlatesPlates5_9_m237_3.sdf' |||| 'BTB08339SC' ||
 * 57.31 || 0 || 43.71 || 0 || -2.79 || -2.59 || 34.39 || 'output_201_400/gold_soln_HF9PlatesPlates5_9_m291_5.sdf' |||| 'BTBG00099SC' ||
 * 57.01 || 6 || 39.53 || 0 || -3.34 || -0.69 || 21.673 || 'output_201_400/gold_soln_HF9PlatesPlates5_9_m328_4.sdf' |||| 'BTB09089SC' ||
 * 57 || 0 || 41.5 || 0 || -0.06 || -4.32 || 25.126 || 'output_1_200/gold_soln_HF9PlatesPlates5_9_m51_3.sdf' |||| 'BTB11079SC' ||
 * 55.95 || 0 || 44.66 || 0 || -5.45 || -0.56 || 36.758 || 'output_201_400/gold_soln_HF9PlatesPlates5_9_m335_3.sdf' |||| 'HTS10322SC' ||
 * 55.94 || 2.03 || 42.18 || 0 || -4.1 || -3.52 || 47.118 || 'output_201_400/gold_soln_HF9PlatesPlates5_9_m257_5.sdf' |||| 'BTB05187SC' ||
 * 55.46 || 0.52 || 40.73 || 0 || -1.06 || -3.89 || 35.272 || 'output_201_400/gold_soln_HF9PlatesPlates5_9_m210_7.sdf' |||| 'SPB01807SC' ||
 * 55.24 || 6 || 41.28 || 0 || -7.51 || 1.25 || 23.629 || 'output_1_200/gold_soln_HF9PlatesPlates5_9_m96_1.sdf' |||| 'KM07005SC' ||
 * 55.22 || 6 || 37.94 || 0 || -2.94 || -6.93 || 11.277 || 'output_1_200/gold_soln_HF9PlatesPlates5_9_m26_3.sdf' |||| 'HTS07322SC' ||
 * 55.46 || 0.52 || 40.73 || 0 || -1.06 || -3.89 || 35.272 || 'output_201_400/gold_soln_HF9PlatesPlates5_9_m210_7.sdf' |||| 'SPB01807SC' ||
 * 55.24 || 6 || 41.28 || 0 || -7.51 || 1.25 || 23.629 || 'output_1_200/gold_soln_HF9PlatesPlates5_9_m96_1.sdf' |||| 'KM07005SC' ||
 * 55.22 || 6 || 37.94 || 0 || -2.94 || -6.93 || 11.277 || 'output_1_200/gold_soln_HF9PlatesPlates5_9_m26_3.sdf' |||| 'HTS07322SC' ||

Best Ranking List for GOLD RUN 2
 * File containing a listing of the fitness of the top-ranked ||  ||   ||   ||   ||
 * individual for each ligand docked in GOLD. || in GOLD. ||  ||   ||   ||   ||   ||   ||
 * Format is: ||  ||   ||   ||   ||   ||   ||   ||   ||
 * Fitness || S(hb_ext) || S(vdw_ext) || S(hb_int) || S(int) || intcor || time || File name |||| Ligand name ||
 * 73.05 || 0.95 || 54.29 || 0 || -2.55 || 10.64 || 434.502 || 'output_1_200/gold_soln_HF9PlatesPlates5_9_m170_3.sdf' |||| 'RJF00101SC' ||
 * 64.86 || 1.85 || 48.11 || 0 || -3.14 || -3.05 || 249.486 || 'output_201_400/gold_soln_HF9PlatesPlates5_9_m257_8.sdf' |||| 'BTB05187SC' ||
 * 62.69 || 1.76 || 47.78 || 0 || -4.77 || 0.44 || 149.452 || 'output_1_200/gold_soln_HF9PlatesPlates5_9_m188_10.sdf |||| ' 'SPB02121SC' ||
 * 62.66 || 1.62 || 47.05 || 0 || -3.65 || -4.44 || 197.409 || 'output_1_200/gold_soln_HF9PlatesPlates5_9_m165_3.sdf' |||| 'RH01522SC' ||
 * 62.36 || 0 || 46.4 || 0 || -1.44 || 0.64 || 35.536 || 'output_1_200/gold_soln_HF9PlatesPlates5_9_m117_3.sdf' |||| 'JFD00064SC' ||
 * 61.64 || 6 || 44.7 || 0 || -5.82 || 1.04 || 68.097 || 'output_1_200/gold_soln_HF9PlatesPlates5_9_m96_1.sdf' |||| 'KM07005SC' ||
 * 60.42 || 1.32 || 45.05 || 0 || -2.83 || -5.12 || 35.733 || 'output_1_200/gold_soln_HF9PlatesPlates5_9_m58_3.sdf' |||| 'DP02063SC' ||
 * 60.31 || 6.82 || 39.64 || 0 || -1.01 || -5.21 || 192.587 || 'output_1_200/gold_soln_HF9PlatesPlates5_9_m45_4.sdf' |||| 'KM04403SC' ||
 * 60.16 || 0.37 || 44.03 || 0 || -0.76 || 22.04 || 85.174 || 'output_1_200/gold_soln_HF9PlatesPlates5_9_m80_2.sdf' |||| 'CD08956SC' ||
 * 59.65 || 0 || 45.73 || 0 || -3.23 || -6.05 || 385.621 || 'output_201_400/gold_soln_HF9PlatesPlates5_9_m392_4.sdf' |||| 'NRB00168SC' ||
 * 59.1 || 6 || 40.58 || 0 || -2.7 || -2.39 || 51.212 || 'output_201_400/gold_soln_HF9PlatesPlates5_9_m237_3.sdf' |||| 'BTB08339SC' ||
 * 60.16 || 0.37 || 44.03 || 0 || -0.76 || 22.04 || 85.174 || 'output_1_200/gold_soln_HF9PlatesPlates5_9_m80_2.sdf' |||| 'CD08956SC' ||
 * 59.65 || 0 || 45.73 || 0 || -3.23 || -6.05 || 385.621 || 'output_201_400/gold_soln_HF9PlatesPlates5_9_m392_4.sdf' |||| 'NRB00168SC' ||
 * 59.1 || 6 || 40.58 || 0 || -2.7 || -2.39 || 51.212 || 'output_201_400/gold_soln_HF9PlatesPlates5_9_m237_3.sdf' |||| 'BTB08339SC' ||

__**WEEK 7**__ Did the Primer Overlap Assembly as I designed a set of forward and reverse primers for PCR for serine threonine phosphotase 2C

This is the DNA sequence for the particular target.



This is the oligonucleotides(primers) that I obtained The protein is serine throenine phosphotase 2C and the strain was made for E.Coli 21



For the particular target there are 24 primers

Also in wet lab I completed the primary and secondary PCR and will do PCR squared this coming week.

__**WEEK 2**__



__Lane 1: skip__ __Lane 2: 100 bp ladder__ __Lane 3: PCR of pGBR22 Tube A__ __Lane 4: PCR of pGBR22 Tube B__ __Lane 5: PCR of pGBR22 Tube C__ __Lane 6: PCR of pGBR22 Tube D__
 * WEEK 2 (PCR WITH pGBR22**)


 * __WEEK 2 (ANALYZING DNA SEQUENCES)__**


 * HUMAN SEQUENCING**


 * NUCLEOTIDE SEQUENCING**






 * COMPARISON OF PURPLE PROTEIN TO pGBR22**
 * a**


 * WEEK 2 RE DIGEST**



Lane 2 1kB ladder Lane 3 uncut pgbr22 plasmid Lane 4 eco R1 Lane 5 PvuII Lane 6 EcoR1 + PvuII


 * WEEK 3 SUBMITTING DNA SEQUENCE**


 * The Requests are entered, your Order Number is: ****72169 **


 * **DNA SEQUENCING REQUEST FORM ** ||

**Grant Account Number ** || ||
 * || **Order No: ** || **72169 ** ||
 * **Contact Name: ** || **arrafi,mohammed ** ||
 * **Email: ** || **mohammedarrafi24@yahoo.com ** ||
 * **Principal Investigator: ** || **beckham, josh ** ||
 * **Department: ** || **Natural Sciences ** ||
 * **Phone: ** || **2106013016 ** ||
 * **<span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 13px;">Service Type: ** || **<span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 13px;">Core_Prep_Template-Only ** ||
 * **<span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 13px;">DNA Prep Method: ** || **<span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 13px;">Qiagen ** ||
 * **<span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 13px;">Todays Date: ** || **<span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 13px;">Sep 14 2011 ** ||  ||   ||   || **<span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 13px;">University of Texas **
 * <span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 13px;">ICMB Core Facilities **
 * <span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 13px;">DNA Sequencing **
 * <span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 13px;">2500 Speedway **
 * <span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 13px;">MBB 1.426L, A4800 **
 * <span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 13px;">Austin, TX 78712 **
 * <span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 13px;"><span class="skype_pnh_print_container">512 475-7844 <span class="skype_pnh_container"><span class="skype_pnh_mark"> begin_of_the_skype_highlighting <span class="skype_pnh_highlighting_inactive_common"><span class="skype_pnh_textarea_span"><span class="skype_pnh_text_span">512 475-7844  <span class="skype_pnh_mark">end_of_the_skype_highlighting  p **
 * <span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 13px;">512 471-2149 fax ** ||  ||
 * **<span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 13px;">26-6815-3050 ** ||
 * <span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 13px;">The order contains 2 Samples. **
 * **<span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 13px;">If submitting 96 well plates, please write ORDER NUMBER on the plate. ** ||


 * || **<span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 13px;">Sample ** || **<span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 13px;">Conc ** || **<span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 13px;">DNA Type ** || **<span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 13px;">Size ** || **<span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 13px;">Primer ** || **<span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 13px;">Primer Conc. ** ||
 * **<span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 13px;">1 ** || **<span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 13px;">MA1 ** || **<span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 13px;">27 ** || **<span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 13px;">pGBR22 ** || **<span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 13px;">4000 ** || **<span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 13px;">M13For-20 ** || **<span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 13px;">5 ** ||
 * **<span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 13px;">2 ** || **<span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 13px;">Ma2 ** || **<span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 13px;">27 ** || **<span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 13px;">ppGBR22 ** || **<span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 13px;">4000 ** || **<span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 13px;">M13Rev-24 ** || **<span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 13px;">5 ** ||

_
 * WEEK 3 (PCR WITH pGBR22 AGAIN)**

Lane 2 1kB ladder Lane 3 sample a Lane 4 sample b Lane 5 sample c Lane 6 sample d


 * WEEK 3 (PCR WITH PNIC BSA4)**



Lane 2: Ladder 100bp Lane 3: 1 in 1000 concentration Lane 4: 1 in 1000 concentration Lane 5: 1 in 1000 concentration

_
 * WEEK 4 (PCR WITH PNIC BSA4)**



__Lane 1: 100bp ladder__ __Lane 2: 1 in 100 concentration__ __Lane 3: 1 in 1000 concentration__ __Lane 4: 1 in 10000 concentration__

__**ALSO IN WEEK 4 COMPLETED THE PYMOL REFRESHER WHICH IS IN GOOGLE DOCS**__


 * WEEK 5** **(PCR WITH PNIC BSA 4)**



WEEK 6 (VIRTUAL SCREENING)

Done with both runs 1 and 2 but didn't have time to analyze them. Well ran run 2 on Thursday but had to rerun it on Friday as there was an error in the run. Also did quite a lot of research on my drug. Re did pnic-bsa4 PCR. Samples are in the fridge i jst have to make a gel and analyze them next week.

Mohammed - put your results in Reverse Chronological order. -- Dr. B