James+T.

Week 13, 14, 15 All 4 libraries (1202 ligands) were screened along with 5 positive controls and 5 random controls for //Listeria monocytogenes//; none of the positive controls made it to the top 120. A random control made it to the top 60. All results were ranked, and the top 30 were submitted to Dr. B and put in my google student folder.
 * Virtual**

YopH activity was measured via enzyme assays. As the amount of enzyme increased, the phosphatase activity also increased. There was no phosphatase activity whatsoever without enzyme. Inhibition assays could not be carried out as the compounds dissolved in DMSO were prepared incorrectly (no compounds were added at all). This lead to 0 inhibition of the enzyme.
 * YopH enzyme and inhibition assays**

Week 11 & 12 Found positive and negative controls but could not get Maestro to function. Told to try again next week when Maestro may be working.
 * Ligand Prep**
 * This is a really intense ROTC week, I didn't get anything else done

SORRY I was dealing with my friend's death last night 9DEC13

Week 9 & 10 The homology model was started but has not been completed; the //Mycobacterium tuburculosis// mPtpB protein structure will be used to construct the homology model.
 * Nice job but could have more results for these couple of weeks. - BN**
 * Homology Model**

Cloning was not succesful, after cohesive end generation and annealing, transformation failed as no colonies were present on the LB+agar+suc plates. This may be attributed to the previous low concentrations of PCR clean up and Midi Prep. More PCR product and pNIC-Bsa4 will be grown up in order for another attempt at cloning.
 * Cloning attempt #2**

The previously mentioned pNIC-Bsa4 cells were put through Midi prep to obtain pure plasmid. The concentration is, again, lower than desired, but there is an obvious increase and decrease in the slope of the concentration graph at 260nm. The plamid will be used for cloning. Good work, captions under images could be better. -UM Week 7 & 8
 * Midi** **Prep**
 * Transformation of competent cells for plasmid prep of pNIC-Bsa4**

pNIC-Bsa4 cells were grown up again and spun down to produce 4 more pellets. They were frozen and stored and will be used for Midi prep in order to obtain pNIC-Bsa4 DNA.


 * PCR^2**
 * Something seems to be up with the new 100bp ladder; they run faster than other ladders and samples.
 * Lane 4 was skipped due to well damage.
 * Faint bands and contamination in this run

Cloning was not succesful, after cohesive end generation and annealing, transformation failed as no colonies were present on the LB+agar+suc plates. This may be attributed to the previous low concentrations of PCR clean up and Midi Prep. More PCR product and pNIC-Bsa4 will be grown up in order for another attempt at cloning.
 * Cloning**

The previously frozen pNIC-Bsa4 pellets had the DNA extracted. The average concentration of two samples after Midi prep came out to be 19.85 ng/ul; this value is extremely low. Thus, cloning will probably not be successful. The DNA sequencing results also did not match to any known genomes when run through BLAST comparisons. Too much DNA may have been lost during Midi prep.
 * Midi Prep**

Good job! What are the last 4 wells in figure 4? Keep up the world class research - Michael T. Week 5 & 6 Grown up overnight, centrifuged, and decanted.
 * pNIC**

//After being left overnight, the LB media mixed with one colony of pNIC and antibiotic appeared cloudy and turbid. This was spun down resulting in 4 pellets which were then stored and frozen.//


 * PyMOL Refresher**

Completed.

PCR cleanup - Completed //The four samples produced one band per lane, thus PCR^2 was almost certainly successful. No contamination bands exist, though light smears appear to have formed above the bands.// //Secondary PCR was successful *(confirmed; PCR^2 succeeded) as evidenced by a single, highly concentrated (bright) band in the lane. There is a slight smear above the band, but the band in lane is overt and bright enough to say that secondary PCR was successful.//
 * PCR**

//Primary PCR was successful as there is a smear in the second lane. There seems to a be a high concentration, as shown by a bright band near the bottom of the smear, which may be oligos not joined together during PCR.//

//Primer dilution was successful as secondary PCR and PCR^2 were successful.//
 * Primer dilution:** Complete

Week 3 & 4 James - very good page! - 092713 - Dr. B
 * Restriction Enzyme Digest**

//This RE digest, though not 100% perfect, worked just as it should. Lanes 2 and 6 had no EcoRI or PvuII and would not have been cut anywhere along the plasmid. The concentration of matter also appear in the same position and the correct position by size in lane. EcoRI would only have cut the plasmid once resulting in one band as in lane 3. PvuII cut the plasmid twice resulting in two bands as shown by lane 4. EcoRI and PvuII cut the plasmid 3 times resulting in three bands as seen in lane 5.//

Forward Primer:5’ TACTTCCAATCC ATGAAGAATTGGGTTAAA 3’ **30** bpGC Content **33.3**% 0 mM Mg2+ Tm **57.2**oC 1.5 mM Mg2+ Tm **65.1** oC 2 mM Mg2+ Tm **65.7**oC 4 mM Mg2+ Tm **67.0** oC 6 mM Mg2+ Tm **67.5**oC Reverse Primer:5’ AAAGCGTACCTGTACTAA CAGTAAAGGTGGATA 3’ **33** bpReverse complement it: 5’ TATCCACCTTTACTGTTAGTACAGGTACGCTTT 3’ **33** bp0 mM Mg2+ Tm **60.4**oC 1.5 mM Mg2+ Tm **68.0**oC 2 mM Mg2+ Tm **68.5**oC 4 mM Mg2+ Tm **69.6**oC 6 mM Mg2+ Tm **70.1**oC GC Content **39.4**%
 * PCR Primer Design Tails**

//Results from analysis of lmon PTP gene of interest.//

//Virtual predictions of how the BsaI enzyme will react with the insert and the full sequence along with virtual PCR images.//

//The gene of interest was analyzed and virtually inserted into the full pNIC-BSA4 sequence and virtually acquired (though not shown). This will hopefully occur in wet lab in order to clone the gene of interest.//

//The results of primary PCR for the GOI appears to have been successful except for the fact that the concentration (bright band) is near the bottom of the ladder when it should be next to the 900 bp marker on the ladder. This could be the result of oligos not coming together. Regardless, this template will be used for secondary PCR.//
 * Primary PCR**

Week 1 & 2 James - good job. Crop your gel images to remove alot of the black. Dr. B 090913

//For the most part, the gel seems to have run successfully since a distinct band does appear in lanes 2-4 for samples A, B, and C, but not so much for lane 5, sample D, which had no DNA. What may be a light band in lane 5 may be due to contamination while loading the other lanes while preparing the gel.//
 * PCR**

 22 oligonucleotides need to be synthesized   1 ATGAAAAATTGGGTTAAAGTTACCGGTGCGGGTGTCCTGTCTGCA 45  2 CCTTCTCTTCAGACTGCGCACCGCAACCACCCAGCAGCAGGGTTGCAGACAGGACACCCG 60  3 GCGCAGTCTGAAGAGAAGGCCGAAGCGAACGTAAAAACCGAGCAGACCCTGAAACCGGGT 60  4 GAGGTCACGGACGTTTACCGCACCTTCCAGTTTGATCTGGCTACCCGGTTTCAGGGTCTG 60  5 GGTAAACGTCCGTGACCTCGGTGGCTACAAAACCACCGATGGTCTCACCATCAAACCGCA 60  6 TGTCAGACAGATTCGCCAGTTCCGCAGAACGGATCAGTTTGTGCGGTTTGATGGTGAGAC 60  7 CTGGCGAATCTGTCTGACAGCGACAAAAAGAAGCTGGTTAACACCTACGACCTGAGCCAC 60  8 CGGTTTGGTCGCAACTTCAGAAGACGTACGGAAGTCTACGATGTGGCTCAGGTCGTAGGT 60  9 TGAAGTTGCGACCAAACCGGACCCGAAACTGACCGACGTTGACTACACCCACGACTCTGT 60  10 TCAGGTCCTGGGTGCTCGTAGAGGTACCGTTGTCCTTCATAACAGAGTCGTGGGTGTAGT 60  11 GAGCACCCAGGACCTGACGGCGTCTCTGGCAAAAATGGACAACCCGGAAACCTTCCTGAT 60  12 GGATAGAAGTTTCGTCGGTGATGAAAGATTTGTTCGCGTTAATCAGGAAGGTTTCCGGGT 60  13 TCACCGACGAAACTTCTATCCAGGCGTACAAAGACTTCTTCGACATCCTGCTGGCCAACC 60  14 CGGTCTTTACCCGCGGTACAGTGCCACAGAACAGAACCGTCTTGGTTGGCCAGCAGGATG 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 15 ACCGCGGGTAAAGACCGTGCAGGTTTCGGCACGGCGCTCGTTCTCTCTGCGCTGGGCGTT 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 16 ATATTTGTTAGACAGCATGTAGTCGTCGATGACGGTGTTCTTGTCAACGCCCAGCGCAGA 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 17 CGACTACATGCTGTCTAACAAATATCGCGCTGACGAAAACAAGAAGGCGATCGAAGCAGT 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 18 GTCATACCGTCGATAACTTTTTTGTTGTCGGTCTTCGCTGCTACTGCTTCGATCGCCTTC 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 19 CAAAAAAGTTATCGACGGTATGACCGCCGTAATGGAAGTTCGTGAATCTTACATCAACGC 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 20 CCATGCTGCCGTACTTCGCATTAATCTCATCGAACGCTGCGTTGATGTAAGATTCACGAA 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 21 CGAAGTACGGCAGCATGGATAATTTCCTCAAAGAAAAACTGGGTCTGACGGACGCTAAAA 60 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;"> 22 TTAGTACAGGTACGCTTTTTTCAGCTGCTCTTTTTTAGCGTCCGTCAGACC 51 <span style="background-color: #ffffff; color: #222222; font-family: arial,sans-serif;">**Fig 1:** Oligo primer sequences optimized for //E. coli// for the //listeria monocytogenes// EGD-e.
 * Oligo Primer Design**

//Oligo primers were successfully designed for the listeria monocytogenes// EGD-e //gene. These were optimized to be grown in E. coli and will be ordered in order to be used in expression of listeria monocytogenes// EGD-e//.//

Week 1

NNNNNNNNNNNNGGGNNNNNGGGCCCGACGTCGCATGCTCCCGGCCGCCATGGCCGCGGGNTTTTAGTGATGGTGATGGT GATGACCGAGCAAAGAGTGGCGTGCAATGGATATTTCACACTGCTCAACAAATGTGTAATCCTTGTTGTGACTGGTTACATCCAGTTTGCGGTCAACATAGTGATACCCTGGCATCCTCACAGGCTTCTTTGCCTTGTAAGTAGATTTGAATTCACACAAATAGTAACCACCTCCTTCCAACTTCAGAGCCATAAAGTTGTTTCCTATCAGCATTCCATCTCGTGCAAAGAGACGCTCAGTGTTGGGTTCCCAGCCCTGTGTCTTCTTCTGCATAACAGGTCCATTGGGAGGAAAGTTCACACCAGAGATTTTGACATTGTAGATGAAACAGTTGCCTTGGATGCTGGAATCATTGCTGACAGTACACACTGCACCATCTTCAAAGTTCATGATCCTCTCCCATGTATATCCCTCAGGGAATGACTGCTTTACATAATCAGGGATGTCTTCAGGGTACTTGGTGAATGGTATGCTTCCGTATTGAGACAGTGGTGATAAAATATCCCAAGCAAATGGCAGAGGTCCACCCTTGGTGACAGTGAGCTTTACCGTCTGCTCCCCCTCGTAAGGCTTTCCTTTTCCATCGCCTTCGACCTCAAAGTAGTGTCCATTGACCGTGCCTGACATATAAACCTTGTANGTCATTTGTTTAGCGATCACACTCATGATATTTCTCCTTCAATCAATCAAAATCACTAGTGCGGCCGCCTGCNNNCGACCATATGGGAGAGCTCCCAACGCGTTGGATGCATAGCTTGAGTATTCTATAGTGTCACCTAAATAGCTTGGNGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCNGAAGCATAAAGTGTAAAGCCTGGNNNNCTAATGANNGAGCTACTCACATTNTNGCGTTGCGNTCNCNNCCGCTTTCNNTNGGAANCNGNNNNCNNCTGCNTNNNANNGNANNNNNNGGNANNGNGNNNNNNNTGGNNNNTNNNNNNNNNNNTNNNNNNNTNNNTGNNNNGNNNTNGNTGNGNNNNNNNNNNNNNNNNNNNNNNGNANNNNNNNNNCNNAANNNNNNNNNNNNNANNNNNNNNNNNNNNNNNNNNNNNNANGCCNNTNNNGGNNNTTNNNNNNANN
 * Submitting to DNA sequencing core:**
 * Fig 1:** The output for the DNA sequencing core for M13F primer for the DNA sequence of the primer witht he base pairs A, T, C, G. N represents base pairs that could not be determined with absolute certainty.

//This particular order had to be run twice through DNA sequencing, however a sequence was obtained and did have comparable results to other organisms but not in the human genome.//


 * Nanodrop:**

//The concentrations obtained from the nanodrop spectrophotometer were consistent, but both were not around 204.5 ng/uL which was the expected concentration. This may be due to the shelf life of the DNA or other environmental factors.//