Ariel+C. 


 * Week 13 and 14**

Enzyme Inhibition Assay





Top scoring ligands from CB306 library
 * Score || S(PLP || ) S(hbond) || S(cho) || S(metal) || DE(clash) || DE(tors) || intcor || Ligand name ||
 * 79.58 || -39.68 || 3.67 || 0 || 5 || 0 || 1.65 || 2.08 || '7676009' ||
 * 78.91 || -39.75 || 0.99 || 0 || 5.97 || 0 || 0.44 || 0.04 || '5282931' ||
 * 78.71 || -42.59 || 2 || 0 || 4.96 || 0 || 0.36 || 0.06 || '7724431' ||
 * 76.06 || -27.41 || 4.44 || 0 || 5.94 || 0 || 0.72 || 0.14 || '7726180' ||
 * 74.22 || -26.69 || 4.35 || 0 || 6 || 0 || 0.79 || 0.06 || '5175515' ||
 * 73.08 || -33.86 || 1.31 || 0 || 5.97 || 0 || 0.28 || 0.01 || '5135469' ||
 * 72.87 || -39.66 || 2.01 || 0 || 4.84 || 0 || 0.95 || 0.02 || '7589277' ||
 * 72.75 || -33.34 || 1.46 || 0 || 5.88 || 0 || 0.13 || 0 || '7814264' ||
 * 72.47 || -43.7 || 0.82 || 0 || 4.66 || 0 || 0.83 || 0 || '7761278' ||
 * 72.26 || -36.97 || 0 || 0 || 6 || 0 || 0.39 || 0.03 || '7397377' ||
 * 71.71 || -15.97 || 3.62 || 0 || 7.97 || 0 || 1.54 || 0.1 || '5192853' ||
 * 71.42 || -17.99 || 2.55 || 0 || 7.85 || 0 || 0.7 || 0.07 || '7842136' ||
 * 70.97 || -16.08 || 2.85 || 0 || 7.91 || 0 || 0.56 || 0.01 || '6433056' ||
 * 70.8 || -37.1 || 2.08 || 0 || 4.96 || 0 || 0.64 || 0.02 || '7437091' ||
 * 70.69 || -38.04 || 1.37 || 0 || 5 || 0 || 0.77 || 0.06 || '6407567' ||
 * 70.31 || -40.41 || 1 || 0 || 4.99 || 0.6 || 1.51 || 0.01 || '7591231' ||
 * 70.26 || -48.73 || 1.85 || 0 || 2.97 || 0 || 0.94 || 0.06 || '7957875' ||
 * 69.44 || -31.47 || 0.9 || 0 || 6 || 0 || 0.37 || 0.03 || '6565654' ||
 * 69.04 || -37.89 || 1.35 || 0 || 4.99 || 0 || 1.46 || 0.05 || '7928256' ||
 * 68.92 || -45.2 || 2.19 || 0 || 2.96 || 0 || 0.6 || 0.61 || '5525637' ||
 * 68.23 || -28.85 || 1.35 || 0 || 5.91 || 0 || 0.07 || 0.02 || '7907769' ||
 * 67.04 || -26.36 || 1.97 || 0 || 5.94 || 0 || 0.43 || 0.01 || '9040855' ||
 * 66.75 || -26.56 || 1.82 || 0 || 5.98 || 0 || 0.6 || 0.02 || '5490779' ||
 * 66.5 || -45.25 || 3.41 || 0 || 2 || 0 || 0.5 || 0.01 || '7679150' ||
 * 66.3 || -31.46 || 0.01 || 0 || 5.99 || 0 || 0.57 || 0.01 || '7278181' ||
 * 66.3 || -31.46 || 0.01 || 0 || 5.99 || 0 || 0.57 || 0.01 || '7278181' ||

Top scoring ligands from InHouseCompounds library
 * Score || S(PLP) || S(hbond) || S(cho) || S(metal) || DE(clash) || DE(tors) || intcor || Ligand name ||
 * 82.59 || -25.16 || 0.02 || 0 || 9.97 || 0 || 1.25 || 0.05 || '5107893' ||
 * 79.06 || -53.29 || 1.1 || 0 || 3.97 || 0 || 0.93 || 0.2 || '7796224' ||
 * 75.39 || -46.82 || 2.16 || 0 || 3.91 || 0.05 || 0.74 || 0.15 || '7706580' ||
 * 73.97 || -47.78 || 2.54 || 0 || 3.56 || 0 || 1.77 || 0.35 || '7752888' ||
 * 73.97 || -47.78 || 2.54 || 0 || 3.56 || 0 || 1.77 || 0.35 || '7752888' ||


 * Week 11 and 12**

Top scoring ligands from NIHClinicalCollection library
 * Score || S(PLP || ) S(hbond) || S(cho) || S(metal) || DE(clash) || DE(tors) || intcor || Ligand name ||
 * 106.64 || -71.3 || 4.74 || 0 || 4 || 0 || 5.92 || 8.14 || 'SAM001246803' ||
 * 93 || -84.87 || 0 || 0 || 2 || 0.05 || 4.22 || 4.63 || 'SAM001246793' ||
 * 87.13 || -56.51 || 7.39 || 0 || 2 || 0 || 1.93 || 0.33 || 'SAM001246840' ||
 * 85.81 || -28.1 || 0.06 || 0 || 9.82 || 0 || 0.71 || 0 || 'SAM001246904' ||
 * 84.97 || -25.06 || 5.51 || 0 || 7.54 || 0 || 1.83 || 0.94 || 'SAM001246552' ||
 * 84.5 || -47.8 || 0.76 || 0 || 5.99 || 0.05 || 0.87 || 0.11 || 'SAM001246721' ||
 * 83.63 || -35.33 || 1 || 0 || 7.96 || 0 || 1.49 || 0.52 || 'SAM001246684' ||
 * 83.26 || -42.97 || 3 || 0 || 5.97 || 0 || 3.48 || 2.4 || SAM001246634' ||
 * 83.04 || -67.94 || 2.27 || 0 || 2 || 0 || 2.06 || 0.41 || 'SAM001247090' ||
 * 82.49 || -33.78 || 1 || 0 || 7.91 || 0 || 2 || 2.19 || 'SAM001246635' ||
 * 82.2 || -70.24 || 1.16 || 0 || 1.94 || 0 || 1.9 || 0.66 || 'SAM001246653' ||
 * 81.17 || -64.98 || 2 || 0 || 2 || 0 || 1.53 || 1.2 || 'SAM001247084' ||
 * 80.99 || -78.98 || 1 || 0 || 0 || 0 || 1.02 || 0.72 || 'SAM001246965' ||
 * 79.38 || -72.12 || 3.84 || 0 || 0 || 0 || 3.47 || 2.2 || 'SAM001246657' ||
 * 79.37 || -58.48 || 4 || 0 || 1.97 || 0 || 1.6 || 0.28 || 'SAM001246618' ||
 * 78.9 || -43.95 || 0.08 || 0 || 5.97 || 0 || 0.58 || 0.01 || 'SAM001246593' ||
 * 78.53 || -70.85 || 2.27 || 0 || 1 || 0 || 2.81 || 0.51 || 'SAM001247096' ||
 * 78.31 || -59.09 || 2.94 || 0 || 2 || 0.08 || 1.13 || 0.74 || 'SAM001246736' ||
 * 78.3 || -54.88 || 0.44 || 0 || 3.99 || 0 || 1.03 || 0.2 || 'SAM001246988' ||
 * 77.98 || -52.12 || 2.82 || 0 || 2.96 || 0 || 0.18 || 0.02 || 'SAM001246720' ||
 * 77.77 || -58.57 || 2.76 || 0 || 1.9 || 0 || 0.78 || 1.1 || 'SAM001246591' ||
 * 77.71 || -55.76 || 4 || 0 || 2 || 0 || 1.29 || 0.36 || 'SAM001246533' ||
 * 77.61 || -50.18 || 2.6 || 0 || 4 || 0 || 2.31 || 0.26 || 'SAM001246985' ||
 * 77.6 || -39.68 || 1.9 || 0 || 6 || 0 || 2.07 || 0.37 || 'SAM001246806' ||
 * 76.1 || -60.27 || 2.27 || 0 || 2 || 0.01 || 2.31 || 1.4 || 'SAM001246534' ||
 * 75.54 || -34.12 || 2.9 || 0 || 5.99 || 0 || 3.15 || 3.06 || 'SAM001246816' ||
 * 75.21 || -71.33 || 2.64 || 0 || 0 || 0 || 3.81 || 3.23 || 'SAM001246783' ||
 * 75.11 || -40.14 || 2.87 || 0 || 4.97 || 0 || 3.29 || 3.13 || 'SAM001246554' ||
 * 74.95 || -40.79 || 0.05 || 0 || 6 || 0 || 2.13 || 2.08 || 'SAM001246625' ||
 * 74.53 || -69 || 2.66 || 0 || 0 || 0 || 1.73 || 0.56 || 'SAM001246671' ||
 * 74.02 || -56.15 || 3.1 || 0 || 1.7 || 0 || 1.29 || 0.96 || 'SAM001246619' ||
 * 73.23 || -58.39 || 1.46 || 0 || 2 || 0 || 0.83 || 0.11 || 'SAM001246970' ||
 * 72.9 || -59.61 || 1.66 || 0 || 1.9 || 0.2 || 1.78 || 0.62 || 'SAM001246672' ||
 * 72.32 || -40.48 || 5.2 || 0 || 2.99 || 0 || 1.28 || 0.88 || 'SAM001246560' ||
 * 72.16 || -58.99 || 1 || 0 || 1.99 || 0 || 1.39 || 0.92 || 'SAM001246658' ||
 * 71.95 || -40.35 || 1.93 || 0.97 || 4.83 || 5.83 || 1.57 || 2.65 || 'SAM001246818' ||
 * 71.71 || -43.01 || 4.34 || 0 || 2.99 || 0 || 1.5 || 0.54 || 'SAM001246528' ||
 * 71.63 || -34.92 || 3 || 0 || 4.94 || 0 || 2.46 || 3 || 'SAM001246530' ||
 * 71.47 || -64.76 || 1.91 || 1 || 0 || 0 || 2.24 || 2.22 || 'SAM001246538' ||
 * 71.1 || -56 || 4.86 || 0 || 1 || 0 || 2.93 || 0.4 || 'SAM001246852' ||
 * 70.74 || -71.19 || 1 || 0 || 0 || 0 || 2.66 || 0.88 || 'SAM001246578' ||
 * 70.67 || -36.25 || 6.18 || 0 || 2.99 || 0 || 1.54 || 1.03 || 'SAM001246767' ||
 * 70.45 || -48.79 || 3.66 || 0 || 1.99 || 0 || 2.17 || 3.1 || 'SAM001246710' ||
 * 70.28 || -57.45 || 1 || 0 || 1.99 || 0 || 1.33 || 0.57 || 'SAM001247060' ||
 * 69.9 || -26.61 || 4.67 || 0 || 5 || 0 || 0.35 || 0.02 || 'SAM001247049' ||
 * 69.9 || -26.61 || 4.67 || 0 || 5 || 0 || 0.35 || 0.02 || 'SAM001247049' ||

Screened control ligand library: In general most positive controls scored higher. Moved on to screening novel ligand libraries
 * Score || S(PLP) || S(hbond) || S(cho) || S(metal) || DE(clash) || DE(tors) || intcor || time || Ligand name ||
 * 67.34 || -33.19 || 2.71 || 0 || 4.81 || 85.37 || 0.3 || 83.16 || 26.101 || '10921447pos3' ||
 * 61.44 || -27.22 || 3.81 || 0 || 3.89 || 192.74 || 1.96 || 196.07 || 25.617 || '10379025pos2' ||
 * 57.99 || -31.28 || 3.03 || 0 || 2.99 || 0 || 0.89 || 1.42 || 41.363 || '10880008pos1' ||
 * 55.78 || -30.97 || 2.35 || 0 || 3 || 0 || 0.19 || 0.14 || 77.403 || '11151757pos7' ||
 * 52.87 || -25.59 || 3.24 || 0 || 2.99 || 0 || 0.24 || 0.14 || 73.074 || '12124355pos10' ||
 * 52.04 || -29.86 || 1.82 || 0 || 2.94 || 0 || 0.52 || 0.13 || 30.427 || '11031366pos4' ||
 * 48.99 || -52.22 || 0 || 0 || 0 || 0 || 1.69 || 0.16 || 48.712 || '36690577neg4' ||
 * 48.07 || -21.85 || 2.76 || 0 || 3 || 0 || 0.09 || 0.11 || 10.051 || '11183084pos5' ||
 * 47.7 || -48.29 || 0 || 0 || 0 || 0 || 0.34 || 0.08 || 40.811 || '28082271neg3' ||
 * 46.8 || -47.88 || 0 || 0 || 0 || 0 || 0.54 || 0 || 29.708 || '17399864neg2' ||
 * 42.81 || -20.26 || 1.98 || 0 || 3 || 0 || 0.74 || 0.11 || 24.961 || 20262261' ||
 * 42.43 || -30.17 || 1.52 || 0.71 || 1 || 0 || 0.22 || 0 || 8.522 || '16654126neg1.5' ||
 * 42.01 || -17.76 || 2.21 || 0 || 3 || 0 || 0.57 || 0.78 || 13.401 || '2244neg1' ||
 * 40.7 || -14.61 || 2.94 || 0 || 3 || 0 || 0.43 || 0.13 || 33.274 || '10877388pos9' ||
 * 37.48 || -10.86 || 2.87 || 0 || 3 || 0 || 0 || 0 || 21.578 || '13818pos6' ||
 * 34.89 || -9.09 || 2.6 || 0 || 3 || 0 || 0 || 0 || 17.615 || '197063pos11' ||

10/31/13 Found active site in homology model, placed dummy ligand in approximate location
 * Week 9 and 10**



Fig: Homology model made with ICM from template 1M63_A, shown as cartoon, with active site shown as sticks colored by element with green carbons, and dummy ligand glycerol shown as yellow sticks

10/29/13-10/30/13 Started Virtual Screening on target protocol Found control ligands, ran LigPrep on them Fig. Positive and negative control ligand CID and properties

10/26/13 Positive clone obtained from colony 8, identified after DNA sequencing and Blastn Fig. Blastn results of aligning DNA sequencing results of colony 8 and template gene Pvivax STPP2b, 1695bp

10/22/13-10/25/13 Created Homology model for target P.vivax STPP2b using template 1M63_chainA and ICM Fig: Homology model made with ICM from template 1M63_A, shown as cartoon

Nice work on keeping your data updated. Try to include DNA sizes in your caption and also where exactly are you with virtual work? Thank you. -Max 10/21/13 10/18/13 A master plate was made from nine of these colonies that grew and 5ml cultures were grown up the night before and midiprepped. All nine samples were nanodropped then sent to DNA sequencing to check which were positive clones.
 * Week 7 and 8**

Figs 1, 2: Nanodrop measurements of the second and third colonies from master plate after midiprep. Measurements from all nine samples were taken, and concentrations ranged from 50 ng/ul to 110.5 ng/ul

DNA sequencing order number: 96613

10/14/13 The two plates with pNIC-Bsa4 with STPP2b in DH5-alpha had a total of eleven colonies successfully grow.



Fig. DH5-alpha with pNIC-Bsa4 + STPP2b on LB agar+kanamycin+sucrose plates

These eleven colonies should have the recombinant plasmid successfully transformed into them. A master plate was made from nine of these colonies and 5ml cultures were grown up overnight and midiprepped.

10/11/13 pNIC-Bsa4 was cut with restriction enzyme BsaI. The results were PCR cleaned up then run on a gel. Lane 1: Empty Lane 2: 1kb ladder Lane 3: empty Lane 4: pNIC-Bsa4 cut with RE BsaI

The lane with the cut plasmid has two clear bands, showing that the plasmid did cut properly at the sites it was supposed to cut at, and the SacB gene has been cut out of the plasmid, leaving the backbone and ends where our target gene STPP2b can be ligated into.

The rest of the cloning procedure was carried out, and the recombinant plasmid transformed into DH5-alpha competent cells and plated onto LB+kanamycin+sucrose plates and placed in the 37 degree incubator.

10/10/13 After pNIC-Bsa4 in DH5-alpha was grown upn LB media overnight, cells were spun down and midiprepped. Nanodrop image of pNIC-Bsa4 plasmid concentration Fig.: Nanodrop image of pNIC-Bsa4 plasmid concentration

A good concentration of the pNIC vector is evident from the Nanodrop.

10/09/13 Gel of PCR squared reaction Lane2: 1kb ladder Lane3: secondary PCR Lane 4-7: PCR squared reaction (annealing temp. 59 degrees)

Clear bands were seen in the PCR squared reaction, and PCR cleanup was performed on the results. Moving forward to cloning.


 * Week 5 and 6**
 * Good data but try to add more detail into your caption and analysis. Thank you. -Max 10/07/2013
 * 10/03/13**
 * Gel of PCR squared reaction, changed annealing temperature to 59 degrees**
 * Lane 1: 1kb ladder**
 * Lane 2-4: PCR squared reaction**


 * Bands are still too faint. Will compare conditions with group member and troubleshoot.**


 * 9/27/13**
 * Gel of PCR squared reaction**
 * Lane 1: 1kb ladder**
 * Lane 2-5: PCR squared product**


 * Very faint bands visible at proper gene length, particularly in lane 3. Redo PCR squared with annealing temperature of 59 degrees celsius that Anita used which worked.**

Week 3 and 4
 * Ariel - ok keep moving forward with your cloning work. We want to get a clone soon. Dr. B 100113**
 * 9/20/13**
 * Nanodrop of elute: pNIC-Bsa4 in DH5-alpha after midiprep.**
 * Negative yield, pNIC cultures have to be regrown and midiprepped**


 * 9/13/13**
 * Lane 1: empty**
 * Lane 2: 1 kb ladder**
 * Lane 3: Secondary PCR**
 * Lane 4-7: Anita's PCR squared**


 * 9/12/13**
 * Gel of overlap PCR**
 * Lane 1: 1kb ladder**
 * Lane 2: Secondary PCR**
 * Lane 3: Anita's secondary PCR**


 * 9/11/13**
 * Gel of Overlap PCR for STPP2b**
 * Lane 1: 1kb ladder**
 * Lane 2: My primary PCR**
 * Lane 3: Secondary PCR (option A)**
 * Lane 4: Secondary PCR (option B)**
 * Lane 5: Anita's secondary PCR**

Week 1 and 2** Ariel - good. Also work on making some pNIC-bsa4 Dr .B 090913 Gel of Overlap PCR for STPP2b Lane 1: empty Lane 2: 100 bp ladder Lane 3: primary PCR with Oligo mix Lane 4: secondary PCR with first and last oligo Lane 5: secondary PCR with for- and rev- tail primers

Neither secondary PCR were successful and the smear from primary PCR is very faint.

Oligonucleotides for target P. vivax STPP2b from DNAWorks: code 1 ATGGAGCCACTCCCTAACCCGAAAAATGACCGTCAGGTTAAAGACGTTGAACCGCCAC 58 2 ACCGTTCGGGTAGAGCAGTTCCAGAGAGAGCGGTTTCGCCGGTGGCGGTTCAACGTCTTT 60 3 CTGCTCTACCCGAACGGTACTGACGAACCACCGGACTATAAAGCTCTGCGTGACCATCTG 60 4 GATGTCCAGACAGTCCTCCTTGCGGATACGGCCTTCCTTCTTCAGATGGTCACGCAGAGC 60 5 AGGAGGACTGTCTGGACATCATCAAACGTGTTATCGACATTGTTTCCAACGAACCTAACC 60 6 GTCACCGACGATGGTAATCGGATCCTGCAGACGGAGCAGGTTAGGTTCGTTGGAAACAAT 60 7 CGATTACCATCGTCGGTGACATCCATGGTCAATACTACGATTTCCTCAAACTGCTGGAAG 60 8 CAGGAACAGAAACTGGGTGTTGTCCGGGTTGCCACCAACTTCCAGCAGTTTGAGGAAATC 60 9 AACACCCAGTTTCTGTTCCTGGGTGATTACGTCGATCGTGGCTCTTTCTCTATCGAAGTA 60 10 GCGGGAAATTGATTTTCAGGGCGTACAGGAGGAGCAGTACTTCGATAGAGAAAGAGCCAC 60 11 CCCTGAAAATCAATTTCCCGCACAAAATCTGGCTCATCCGCGGTAACCACGAATGCCGTC 60 12 GTATTCACACTCGTCACGAAAATTGAAAAAAGAGGTCATCTGACGGCATTCGTGGTTACC 60 13 ATTTTCGTGACGAGTGTGAATACAAATACGACATGGTTGTTTACTACGCGTTCATGGAAT 60 14 GCCATTGATAACCGCAGACAGCGGGATGGTGTCGAAAGATTCCATGAACGCGTAGTAAAC 60 15 TGTCTGCGGTTATCAATGGCAAATTCCTCGCTGTGCACGGTGGTCTGTCCCCGCAGCTCG 60 16 CGGCTCCTGGAAGCGGGTAAAAGAGCAGATTTGGTTCAGCAGAACGAGCTGCGGGGACAG 60 17 CCGCTTCCAGGAGCCGCCTCGTAGCGGTATCTTCTGCGATATCCTGTGGGCCGACCCAAT 60 18 GTAAGATTCGGTTTGGATAGTGTGTTCTTCTTTATCTTCGTCAATTGGGTCGGCCCACAG 60 19 CACACTATCCAAACCGAATCTTACTTTCCTAACGACATCCGTGGTTGTAGCTATTTCTTC 60 20 CCGTTTTTTTCGAGGAAGGTGGTCGCCGCATTATAACCGAAGAAATAGCTACAACCACGG 60 21 CACCTTCCTCGAAAAAAACGGTCTGCTGTCTATTATCCGTGCGCATGAAGCGCAACTCGA 60 22 GGAAAACCGGTCTTCAGGTTGGTTTGGTGCATTTTGTAACCTTCGAGTTGCGCTTCATGC 60 23 AACCTGAAGACCGGTTTTCCAATCGTTATCACCATCTTTTCTGCGCCGAACTATTGTGAC 60 24 TTAGAATCGAATTTGAGAACCGCACCTTTGTTGTTGTAAACGTCACAATAGTTCGGCGCA 60 25 GCGGTTCTCAAATTCGATTCTAATACCCTGAATATCCAGCAATTTAGCTTCTCTCCACAC 60 26 CAGGTGAAGAGGTTCATAAAGTTCGGGAGGTGATATGGGTGTGGAGAGAAGCTAAATTGC 60 27 GAACTTTATGAACCTCTTCACCTGGTCTCTGCCGTTCGTCTCTGAGAAAGTTACCGAAAT 60 28 AGACTGGTTAACAGAAGAGTTCAGAATGCAGTACAGCATTTCGGTAACTTTCTCAGAGAC 60 29 TGAACTCTTCTGTTAACCAGTCTGACGAGGGTGTTAAGGATGTAGTTCTCCCAGCGGAGG 60 30 AGTTTAACGTTGTTCTCCTCGATATAAGAGATGATCTGGAGTACCTCCGCTGGGAGAACT 60 31 TCGAGGAGAACAACGTTAAACTGGAGGGTATGACCCTCAGCGGCGGTGGTGGCGCAACGG 60 32 GAGAAGACGGGGAACCCTCGGTAGCACCGGTAGACGCTGCACCAGCCGTTGCGCCACCAC 60 33 AGGGTTCCCCGTCTTCTCAGCGTAAGGAAGCGCTCTTTAAGGAGGGCTGCTTCCACTCTG 60 34 CAGGAGAGGTGGTACCCAGTGCACCGCCTTCTTTGGACGCACCAGAGTGGAAGCAGCCCT 60 35 CTGGGTACCACCTCTCCTGCGGCAGCTACCGCGACTACTCAGCAAATGGCTGCCCAAGGT 60 36 AGACGCCTGCGCGTCATCGGTATGCAGGTGCGCTGGCTGTTCACCTTGGGCAGCCATTTG 60 37 GACGCGCAGGCGTCTAAAGAACGTAGCGACGCGCTGCGTAAAAAAGTCCAGAGCGTCGGT 60 38 GTTCGTTTTCTTTGCGCAGAGTACGGAATACACGCATCAGACGACCGACGCTCTGGACTT 60 39 TCTGCGCAAAGAAAACGAACTCATCGTTCAACTGAAAGGTTGCTCTCCGGGCTATCGTAT 60 40 TCTCCAGACCCTCTTTACCTTGGAGCAGCAGGCCAACAGGAATACGATAGCCCGGAGAGC 60 41 AAGGTAAAGAGGGTCTGGAGAATGAACTGGAAAAATTCACCAAAGCGAAACAGATTGACT 60 42 TTATTCGTTTGGCGGACGTTTTTCGTTAATAGAGTCAATCTGTTTCGCTTTGG 53 code

Fall '13 start

Ariel - good job. - Dr. B 071713

Things To Do: Submit to DNA Sequencing Analyze DNA Sequence Exercise PCR of pGBR22 Red OR Green PCR PCR of pNIC-Bsa4 with pLIC-for and pLIC-rev primers

7/17/13 pNIC PCR gel Lane 1: 100bp ladder Lane 2: Sample 1 (Lowest concentration template) Lane 3: Sample 3 (Highest concentration template)* Lane 4: Sample 2 (Next highest concentration template)* Lane 5: Sample 4 (0 ng concentration template) Lane 6: SKIP

*mistakenly loaded in non-sequential order
7/16/13 mCherry PCR gel Lane 1: SKIP Lane 2: 100bp ladder Lane 3: Sample 1 (0.0112 ng template with for primer VDSR1/rev primer VDSR2) Lane 4: Sample 2 (0.112 ng template with for primer VDSR1/rev primer VDSR2) Lane 5: Sample 3 (1.12 ng template with for primer VDSR1/rev primer VDSR2) Lane 6: Sample 4 (0 ng template with for primer VDSR1/rev primer VDSR2) Lane 7: Sample 5 (0.0112 ng template with M13 for/rev primer) Lane 8: Sample 6 (0.112 ng template with M13 for/rev primer) Lane 9: Sample 7 (1.12 ng template with M13 for/rev primer) Lane 10: Sample 8 (0 ng template with M13 for/rev primer)

Lane 6 (Sample 4) should not have a visible band present in it, and this may be evidence of contamination or some of the sample from Lane 5 moving into Lane 6

7/11/13 Failed Red PCR gel

7/9/13 pGBR22 PCR Lane 1: SKIP Lane 2: 100bp marker Lane 3: Sample A (0.3 ng of template) Lane 4: Sample B (3 ng of template) Lane 5: Sample C (30 ng of template) Lane 6: Sample D (0 ng of template)

7/5/13 Lane1: SKIP Lane2: 1kb DNA ladder Lane3: uncut plasmid Lane4: EcoRI Digestion Reaction Lane5: PvuII Digestion Reaction Lane6: EcoRI + PvuII Digestion Reaction