ResearchPage+-+Michael

=__Michael's Research__= toc =__sbPSTP (2CM1) Research__:=
 * ==Mycobacterium Tuberculosis H37Rv==

> ===PCR: Primer Overlap===
 * ==Week 12:==
 * ===FPLC Ahh! - Embed the image instead of linking to it....... Dr. B===
 * [[file:20111117_MichaleEaston_FPLCresult_PSTP.bmp]]
 * ===Protein Characterization===
 * [[image:IMG_20111118_144151.jpg width="560" height="417"]]
 * ==Week 11:==
 * ===Protein Purification===
 * [[image:ma986_elution1&2.jpg width="552" height="342"]]
 * ==Week 10:==
 * ===Protein Expression===
 * Lysed the pellets with lysis reagent and spun down the contents
 * ===PCR: Primer Design===
 * Secondary PCR for 2nd and 3rd attempt, running Gel Sunday
 * ==Week 9:==
 * ===Protein Expression===
 * [[image:mae986_pellet.png width="560" height="417" caption="Pellets are highlighted"]]
 * Pellet weight: .97 +1.59 = **2.56g**
 * [[image:mae986_graph.png]]
 * At OD600
 * **1st** measure **12:00** 0.0628265235129
 * **2nd** measure **12:10** 0.146219267443
 * **3rd** measure **2:20** 0.391527989213
 * ==Week 8:==
 * ===PCR: Primer Overlap===
 * Made new Primary PCR after repeated failures of last week
 * ===Virtual Screening===
 * ==Week 7:==
 * ===PCR: Primer Overlap===
 * **L**ane 1: **100bp Ladder;** Lane 2: **Secondary PCR**
 * [[image:mae986_PCR_2cm1secondaryRun2.JPG width="560" height="571" caption="Primer overlap for sbPSTP, comparing to a 100 bp ladder. 2nd run of secondary PCR failed."]]
 * ==Week 6:==
 * **Lane 1:** 100bp Ladder, **Lane 2:** Primary PCR; **Lane 3:** Secondary PCR
 * [[image:mae986_PCR_2cm1primary_secondary.JPG width="560" height="545" caption="Primer overlap for sbPSTP, comparing to a 100 bp ladder. The primary PCR looks succesful as there should be a smear; however the secondary PCR has extra bands, where it should only have a band at ~700bp"]]
 * ==Week 5:==
 * ===PCR Primer Design for Primer Overlap Assembly PCR===
 * Protein: sbPSTP
 * Organism: Mycobacterium Tuberculosis PSTP
 * code format="term"


 * Helix Systems -- Center for Information Technology              |
 * http://helix.nih.gov                             |
 * National Institutes of Health, Department of Health and Human Services    |
 * DNAWorks Web Site: http://helixweb.nih.gov/dnaworks             |
 * DNAWorks Web Site: http://helixweb.nih.gov/dnaworks             |


 * Send all correspondence to webtools@helix.nih.gov              |
 * Send all correspondence to webtools@helix.nih.gov              |

Job started on 10/02/2011 at 19:39:44 Job name: ME100211sbPSTP Output will be sent to maeaston25@gmail.com Starting  1 trial... PARAMETERS FOR TRIAL 1 Total Size Of Gene ......... 723 nt                                      Protein Residues ........... 241                                         Mutatable Residues ......... 235                                         Fixed Nucleotides .......... 18 nt                                       Oligo Size ................. 60 nt                                       Annealing Temp ............. 62 +/- 1*C Oligo Concentration ........ 1.00E-7 M                                   Sodium Concentration ....... 5.00E-2 M                                   Mg2+ Concentration ......... 2.00E-3 M                                   Codon Frequency Threshold .. 10%                                         Repeat Threshold ........... 8 nt                                        Mispriming Threshold ....... 8/18 (6 exact) nt                    100 optimization rounds, best =     3.436 Rep =   6 Mis =   0 The total codon usage score ........... 0.000 The total length score ................ 0.000 The total melting temperature score ... 0.000 The total repeat score ................ 0.000 The total pattern score ............... 0.000 The total mispriming score ............ 0.000 The total AT content score ............ 0.000 The total GC content score ............ 0.000 The total fixed gap score ............. N/A The OVERALL score ...... 0.000 Finished trial  1 : rate =   0.2 min/cyc : time remaining =   0.0 min.                          FINAL SUMMARY FOR   1 SOLUTION

#   Tm   Len  |    Score   TmRange  Short    Long   #Olig  #Repeat #Misprime 1   62    60  |    0.000       1.8      15      60      18       0       0


 * Helix Systems -- Center for Information Technology              |
 * http://helix.nih.gov                             |
 * National Institutes of Health, Department of Health and Human Services    |
 * DNAWorks Web Site: http://helixweb.nih.gov/dnaworks             |
 * DNAWorks Web Site: http://helixweb.nih.gov/dnaworks             |

Finished code

=__First Protocols:__=


 * ==Week 4:==


 * ===Practice PCR: PNIC-bsa4===
 * **Lane 1:** 100bp Ladder; **Lane 2:** 100bp Ladder; **Lane 3**: 1:10 Dilution; **Lane 4:** 1:100 Dilution; **Lane 5:** 1:1000 Dilution; **Lane 6:** Control
 * [[image:mae986_pcr2_practice_run2.JPG width="560" height="558" caption="No Yield, possible contamination of lane 3"]]
 * **Lane 1:** Empty; **Lane 2:** 100bp Ladder; **Lane 3**: 1:100 Dilution; **Lane 4:** 1:1000 Dilution; **Lane 5:** 1:10000 Dilution; **Lane 6:** Control
 * [[image:mae986_pcr2_practice_run1.JPG width="558" height="517" caption="No Yield"]]
 * ===Virtual Screening Refresher:===
 * 2nd Run submitted, still waiting to be run


 * ==Week 3:==


 * ===REDigest===
 * **Lane 1:** empty; **Lane 2:** 1kb Ladder; **Lane 3:** uncut plasmid; **Lane 4:** EcoRI; **Lane 5:** PvuII; **Lane 6:** EcoRI & PvuII
 * [[image:mae986_practice_redigest_1.JPG width="560" height="586" caption="PvuII cut the plasmid pGBR22 properly in both lanes 5 and 6. EcoRI failed to cut in either lane 4 or 6 and causing multimer bands to form in lane 4."]]


 * ==Week 2:==


 * ===Practice protocol for PCR===
 * [[image:mae986_pcr_practice_run1.JPG caption="1st attempt of pcr with no results"]]
 * Lane 1: Empty; Lane 2: 100bp Ladder; Lane 3: 1ul 1:1000; Lane 4: 10ul 1:1000; Lane 5: 10ul 1:100; Lane 7: overflow
 * [[image:mae986_pcr_practice_run2.JPG width="560" height="583" caption="2nd run of PCR with pGBR22 template showing ~1000bp. Output is stronger in lanes 4&5 where the intial concentration of template is higher. lane 6 yielded nothing, being the control. Lane 7 is possible over flow from the other lanes."]]


 * ==Week 1:==
 * ===DNA Sequencing===

1. pGBR22 M13F NNNNNNNNNGTATACGACTCACTATAGGGCGAATTGGGCCCGACGTCGCATGCTCCCGGCCGCCATGGCCGCGGGATTTTAGTGATGGTGATGGTGATGACCGAGCAAAGAGTGGCGTGCAA TGGATATTTCACACTGCTCAACAAATGTGTAATCCTTGTTGTGACTGGTTACATCCAGTTTGCGGTCAACATAGTGATACCCTGGCATCCTCACAGGCTTCTTTGCCTTGTAAGTAGATTTG AATTCACACAAATAGTAACCACCTCCTTCCAACTTCAGAGCCATAAAGTTGTTTCCTATCAGCATTCCATCTCGTGCAAAGAGACGCTCAGTGTTGGGTTCCCAGCCCTGTGTCTTCTTCTG CATAACAGGTCCATTGGGAGGAAAGTTCACACCAGAGATTTTGACATTGTAGATGAAACAGTTGCCTTGGATGCTGGAATCATTGCTGACAGTACACACTGCACCATCTTCAAAGTTCATGA TCCTCTCCCATGTATATCCCTCAGGGAATGACTGCTTTACATAATCAGGGATGTCTCAGGGTACTTGGTGAATGGTATGCTTCCGTATTGAGACAGTGGTGATAAAATATCCCAAGCAAATG GCAGAGGTCCACCCTTGGTGACAGTGAGCTTTACCGTCTGCTCCCCCTCGTAAGGCTTTCCTTTTCCATCGCCTTCGACCTCAAAGTAGTGTCCATTGACCGTGCCTGACATATAAACCTTG TAGGTCATTTGTTTAGCGATCACACTCATGATATTTCTCCTTCAATCAATCAAAATCACTAGTGCGGCCGCCTGCAGGTCGACCATATGGGAGAGCTCCCAACGCGTTGGATGCATAGCTTG AGTATTCTATAGTGTCACCTAAATAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGC CTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCNGCTTTCCAGTNGGAAACCTGTCNTGCCAGCTGCATNANGAATCGNCANNNNNNGGGNNANGNNGNTT GCGTNTNGGNNCTNNTNNNCTNCNTNNNNNNNTGACTCGCTGNNNNNGNCNNNGNTGNNNNNNNNNNNNCANNTNNNTNNNNGNNNNNCGNNNNNNNNNNNCNGGNNNNNCNGAAANANNNN NNNNNNNNANNNNNNNNNNNNNNNNCNNNAANNNNNNNNNNNTNGNNTTTNNNNNNNNN NCNCCCCCNNNNNN

2.pGBR22 M13R NNNNNNNNNNTGNNNATAGAATACTCAAGCTATGCATCCAACGCGTTGGGAGCTCTCCCATATGGTCGACCTGCAGGCGGCCGCACTAGTGATTTTGATTGATTGAAGGAGAAATATCATGA GTGTGATCGCTAAACAAATGACCTACAAGGTTTATATGTCAGGCACGGTCAATGGACACTACTTTGAGGTCGAAGGCGATGGAAAAGGAAAGCCTTACGAGGGGGAGCAGACGGTAAAGCTC ACTGTCACCAAGGGTGGACCTCTGCCATTTGCTTGGGATATTTTATCACCACTGTCTCAATACGGAAGCATACCATTCACCAAGTACCCTGAAGACATCCCTGATTATGTAAAGCAGTCATT CCCTGAGGGATATACATGGGAGAGGATCATGAACTTTGAAGATGGTGCAGTGTGTACTGTCAGCAATGATTCCAGCATCCAAGGCAACTGTTTCATCTACAATGTCAAAATCTCTGGTGTGA ACTTTCCTCCCAATGGACCTGTTATGCAGAAGAAGACACAGGGCTGGGAACCCAACACTGAGCGTCTCTTTGCACGAGATGGAATGCTGATAGGAAACAACTTTATGGCTCTGAAGTTGGAA GGAGGTGGTTACTATTTGTGTGAATTCAAATCTACTTACAAGGCAAAGAAGCCTGTGAGGATGCCAGGGTATCACTATGTTGACCGCAAACTGGATGTAACCAGTCACAACAAGGATTACAC ATTTGTTGAGCAGTGTGAAATATCCATTGCACGCCACTCTTTGCTCGGTCATCACCATCACCATCACTAAAATCCCGCGGCCATGGCGGCCGGGAGCATGCGACGTCGGGCCCAATTCGCCC TATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGNGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAG CGAANAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGNNAATGGNCGCNNCCTGTAGCGNNGCATNNCGCGGNNGGGNGNNGNNGNNCNNNCANCGNGACNCTACNNN NNNNNNCCNNNNNCCGNNNCTTTCGNTTNNNTNCNNTTNNNNGCCNCNTNNNNNTTNCCCNNNANNTNNAANNGGGNNNCNTTNGNNNNCNANTNNNNNTNNGNNNNNNACNNNNAANCNNN NNNNNGNNNNNNNNNNNNNGNNNNNNNCCCNNNNNNANGNNNTNNCCNNNNNNNCNNTNNNNNNN

= The more you learn: =

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