Antonio+G.

Fall 2013 - Week 10 & 11 __**Cell Lysis, Protein Purification and Characterization (10/13/13)**__  The DH5a cells containing the DHFR protein were lysed with a sonicator. The enzyme was purified using a Ni-NTA resin column, with a buffer of 300mM imidazole as the elution solution. A PAGE gel was run to verify proper purification of the protein, it was stained, and left to destain overnight.  __**Large-Scale Expression (10/09/13)**__ The small overnight cultures were grown in a larger volume, activated with IPTG, and centrifuged for protein harvesting. The pellet with a weight of 2.45g, was stored in the -80°C freezer.  __**Growth and Small Overnight Culture (10/08/13)**__  Many colonies were observed growing on the plate of transformed cells. Two small overnight cultures were created using two colonies from the transformation plate (10/07/13), and allowed to grow overnight at 37ºC.  __**Transformation of DH5a (10/07/13)**__ <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Pre-isolated plasmids containing the //E. coli// DHFR gene were transformed into competent DH5a cells. The cells were plated on an LB+Kan plate, and allowed to grow overnight at 37ºC. <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">__**Surrogate Enzyme (10/05/13)**__ <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Attention was switched from the DHFR-TS of //L. major// drug target, to the surrogate enzyme target DHFR of //E. coli// so as to make positive forward progress in lab.

<span style="font-family: 'Times New Roman',Times,serif; font-size: 20px;">Fall 2013 - Week 7 & 8 <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;"><span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">__**Transformed Cell Growth (10/19/13)**__ <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;"><span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Growth of the transformed DH5a cells was observed on the morning of 10/19. Plates were stored at -20ºC until a master plate could be created. <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;"> <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;"><span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">__**Cohesive End Generation, Annealing, and Transformation (10/18/13)**__ <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;"><span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;"><span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Cohesive end generation of PCR² (09/06) and cut pNIC-Bsa4 was performed. This was followed by annealing and transformation into Competent DH5a cells. The transformed //E.coli// was plated onto two Kan+Sucrose plates, and left to incubate overnight at 37º C. __**Virtual Screening of ChemBridge Diversity Library (10/17/13)**__ <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;"><span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">A screening job was submitted for docking of the ChemBridge Diversity Library of 50k compounds in the homology mode active site. <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;"> <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Cut pNIC-Bsa4 plasmid was cleaned using Sigma PCR Clean-Up Kit. Final concentration ~40.0ng/uL <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;"><span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;"> __**Crystal Structure of DHFR-TS (//L. major//) Received (10/15/13)**__ <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;"><span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">A crystal structure for DHFR-TS (//L. major//) was received via email from Dr. David Matthews. This structure will be modified and prepared for all future virtual screening runs. Structure available in student folder (Google Drive). <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;"> <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">__**PCR Squared Performed (10/14/13)**__PCR Squared was performed, and a 1% agarose gel was run. The sample showed too much contamination in the form of multiple unwanted bands. This sample will not be used.
 * Good work Tony. Nice captions and analysis. Thank you. -Max 10/21/13

<span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;"> <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">__**GOLD Validation With Control Library (10/11/2013)**__ <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">A control docking was performed on the 3INV_A homology model in order to validate docking using GOLD. <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">__ **Sequencing Results (10/10/13)** __ <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;"> Sequencing results for three colonies were received. None were better than 10% match against the expected nucleotide sequence. <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;"> <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;"><span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">__**Sequencing Request (10/08/13)**__ <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;"><span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Overnight cultures of the 3 colonies were spun down, mini-prepped, and sent for sequencing using pLIC-For and pLIC-Rev as primers. <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;"> <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;"><span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">__**Small Overnight Cultures, and Midi-Prep (10/07/13)**__ <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;"><span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">The 3 growing colonies were used to begin small overnight culture. <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;"> <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;"><span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">The pNIC-Bsa4 pellet (10/06) was midi-prepped, for a final concentration of ~58ng/uL. <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;"> <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;"><span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">__ **Colony Growth, and Spin-Down (10/06/13)** __ <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;"><span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Three colonies were observed to be growing on the overnight plate. It was left overnight again to provide opportunity for further growth. <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;"> <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;"><span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">pNIC-Bsa4 culture was spun down, decanted, and stored at -20ºC until mid-prep was possible. <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;"> <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">__**Cloning and New pNIC Colony (10/05/13)**__ <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Cohesive end generation was performed on both pNIC and the gel-extracted PCR Squared product, following cloning protocol. The transformed cells were plated on a Kan+Sucrose agar overnight. <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;"> <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Another starter culture of pNIC-Bsa4 was created in 160mL of LB. Left in the shaking incubator overnight.

<span style="font-family: 'Times New Roman',Times,serif; font-size: 20px;">Fall 2013 - Week 5 & 6 <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">**Nice Job maintaining your page for these weeks. Keep up the good work. Try to add more information to your nanodrop captions. Thank you.** <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">**-Max 10/07/2013** <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">__**Second Round of Mass PCR Squared & Midi-Prep of pNIC (10/03-10/04/13)**__ <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">On 10/03, four PCR Squared reactions were performed using the 2º PCR product from 09/25. On 10/04, four 1% agarose gels with ten wells each were created and placed into four separate electrophoresis rigs. The PCR Squared product was combined and prepped with loading gel. 27uL of this solution was loaded into each well. After running, the bands at ~1,500kB were excised and Gel Extraction was performed using a GenElute Kit. Despite the careful attention to detail, and troubleshooting based on 10/20/13 attempt, the concentration was ~20ng/uL. <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">On 10/04, the overnight culture pNIC-Bsa4 plasmid was midi-prepped, for a final concentration of ~6 ng/uL. The low concentration was most likely a result of a technical error during the procedure.





<span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">__**Overnight Culture of pNIC-Bsa4 (10/03/13)**__ <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">A colony from the cloned pNIC-Bsa4 was grown in 160mL of LB overnight in the shaking incubator. <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">__**Cloning of pNIC-Bsa4 Into Competent Cells & Docking NADP Into Homology Model Using ICM (10/02/13)**__ <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">pNIC-Bsa4 plasmid was cloned into competent DH5-a cells and plated on a Kan/Agar petri dish. Colonies were allowed to grow overnight. <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Protocol for docking a ligand into the active site of a .PDB structure using ICM was followed to dock NADPH into the active site of homology model 3invA. Attempts to run a docking script were unsuccessful. Help from the Research Educator was sought.



<span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">__**Docking Attempt of NADP Into Homology Model Using Hermes/GOLD (10/01/13)**__ <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Docking of NADPH into the the homology model was attempted on the early morning of 10/01, only to discover that the license for the Hermes program had expired the day before. Help from the Research Educator was sought. <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">__**Ligand Preparation of Control Library (09/29/13)**__ <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">The control ligands from 09/27 were concatenated into a single .sdf file. ICM was used to ligand-prep the library for docking. <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">__**Creating a Control Library (09/27/13)**__ <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">A positive and negative control library of 17 ligands was created using protocol "ProtocolVirtualScreenYOURTarget_VDS_Fall13_PART1.doc". The library is still to be prepared for docking. All physico-chemical properties of ligands were recorded and made available on the Target Page for DHFR-TS L. major. A list of the chosen control ligands follows:

<span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">1. DHF (Natural Substrate) <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">2. 1CX <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">3. UMP <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">4. C50 <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">5. MTX <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">6. TMQ <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">7. 2CY <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">8. DQ1 <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">9. WRA <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">10. P128 <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">11. P65

**<span style="font-family: 'Times New Roman',Times,serif; font-size: 20px;">Fall 2013 - Week 3 & 4 ** <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">**Tony - great job on Homology and PCR squareds - keep driving fowrard with the cloning - we need to get your clone soon so that you guys can move forward - Dr. B 100113** <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">__**Mass PCR Squared (09/19-20/13)**__ <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">On 09/20, four PCR Squared reactions were performed using the 2º PCR product from 09/02. On 09/21, four 1% agarose gels with six wells each were created and placed into four separate electrophoresis rigs. The PCR Squared product was combined and prepped with loading gel. 50uL of this solution was loaded into the wells of the agarose gels. After running, the bands at ~1,500kB were excised and Gel Extraction was performed using a GenElute Kit. The elution from this extraction (concentration: 77.9 ng/uL) was cleaned using a Sigma PCR CleanUp kit. The low yield of DNA (3.6 ng/uL) resulted in the discarding of the newly-cleaned elution.





<span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">__**Creating a Homology Model (09/18/13)**__ <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Two custom homology model was created for the L. major DHFR-TS enzyme using the ProMol software. PDB entries 3invA and 3irmB were utilized as the template off of which the models were created. The homology structure synthesized using 3invA was the better of the two models, as indicated by the MolProbity score of 2.61. This model will be the basis for initial virtual screening trials.



<span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">__**pNIC-Bsa4 Culture (09/13-14/13)**__ <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">A1 culture of pNIC-Bsa4 was grown in 160mL of LB overnight. The following morning it was determined that no proliferation had occurred - most likely due to the age of the plate from which the colony was taken. <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">__**Swiss Model (09/10/13)**__ <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">A Swiss Model screening was performed on the known L. major DHFR-TS Amino Acid sequence. The program determined that PDB entires 3invA and 3irmB would be suitable for the creation of a homology model.

**<span style="font-family: 'Times New Roman',Times,serif; font-size: 20px;">Fall 2013 - Week 1 & 2 ** <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">**Good - Dr. B 090913** <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">__**PCR Squared (09/06/13)**__ <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">PCR² was performed on the 2º PCR product from 09/02/13. A gel was run to confirm that the band was amplified and at the correct nucleotide length

<span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">__**Primer Re-Ordering, Primary PCR, Secondary PCR (09/02/13)**__ <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">An new oligo mix was created from primers D4-G5 from primer box B. Primary PCR was performed using the new oligo mix. Secondary PCR was performed using custom primers ordered on 07/03/13. A band was present at ~1500 nucleotides - corresponding with the target gene length of 1590 nucleotides. Samples were stored at -20ºC until PCR² was ready to be performed.

<span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">__**Week** **8**__

<span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Sequencing Results from Master Plate #2 (07/24/13) <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">The Colony 9 Reverse sequence confirmed the fact that there were two deletions near the second half of the gene. Master Plate #2 results showed only Colony 18 as a promising plasmid. However, there was one point mutation that was not silent.

<span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Colony 9 Reverse Sequence (07/23/13) <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">A sequencing request was placed for Colony 9 using pLic-Rev as a final confirmation that it was not a positive clone.

<span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Blast of Colony 9 Plasmid & Miniprep #2 (07/22/13) <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">CORE Sequences (Forward and Reverse) for Colony 9 were received. The Reverse Sequence was "reverse complemented" and joined with the forward sequence at the points where overlap occurred. The entire sequence was blasted against the known gene. The Blast returned a 100% Query Coverage and a 99% Identity Match. There were two areas of deletion and one point mutation. The clone was negative.

<span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Master plate tubes from 07/19/13 were Miniprepped. Concentrations ranged from 34.45-89.50 ng/uL. Samples of the 6 colonies with the highest concentrations were sent for sequencing using pLIC-For as the primer (Colonies 21, 19, 18, 15, 17, and 24). Secondary samples of colonies 21, and 19 were sent with pLIC-Rev as the primer.



<span style="font-family: 'Times New Roman',Times,serif; font-size: 20px;">Week 7 <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Harvesting and Colony 9 Digest (07/19/13) <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">The culture tubes were spun down, and the supernatant was poured out. The tubes were stored in the -20ºC freezer. <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">A digest of only Colony 9 plasmid was performed using XmnI and a higher concentration of plasmid in order to visualize the smallest band. No valuable information was obtained from the digest gel.



<span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Sequencing Results & Master Plate 2 (07/18/13) <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">The resulting CORE sequences were blasted against the known gene sequence. Colony 9 resulted in the highest query coverage and identity match (47% Coverage, 96% Identity). An overnight culture of colony 9 was grown overnight. A second master plate and corresponding starter culture tubes were created.



<span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Master Plate Digest (07/17/13) <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">A digest of the transformed pNIC-Bsa4 plasmid was performed using AcuI as the restriction enzyme. A plasmid containing at least some portion of the gene would result in 3 bands when digested. A non-cloned plasmid would result in 2 bands. All plasmids only showed two bands, however this may have likely been a result of a low amount of plasmid, not allowing the smallest band to be seen.



<span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Master Plate Sequencing (07/16/13) <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Samples of the 6 colonies with the highest concentrations were sent for sequencing using pLIC-For as the primer (Colonies 3, 2, 6, 1, 9, and 10). Secondary samples of colonies 3, and 2 were sent with pLIC-Rev as the primer.

<span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Presentation (07/15/13) <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Presented during weekly meeting about Journal Club article. No lab time.

__<span style="font-family: 'Times New Roman',Times,serif; font-size: 20px;">Week 6 __

<span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Harvesting & Miniprep (07/12/13) <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">The Master Plate tubes were spun down and purified using the Miniprep Kit from Sigma. Concentrations ranged from 19.1-46.6 ng/uL.

<span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Master Plate and Colony & Overnight Culture (07/12/13) <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">A pair of master plates was created in the evening. 12 tubes corresponding to the master plate squares were prepared and left in the 37ºC shaking incubator overnight.





<span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Transformation #2 (07/11/13) <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">No growth occurred overnight, so plates were discarded and transformation was attempted again.



<span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Transformation #1 (07/10/13) <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Cohesive end generation of PCR² and cut pNIC-Bsa4 was performed. This was followed by annealing and transformation into Competent DH5a cells. The transformed bacteria was plated onto two Kan+Sucrose plates, and left to incubate overnight at 37º C.

<span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">PCR Cleanup, pNIC Digest (07/09/13) <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">PCR² product was cleaned using Sigma PCR Cleanup Kit. The product had an average concentration of 118.6 ng/uL.



<span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">pNIC-Bsa4 was digested using BsaI-HF, then purified using a Cleanup Kit. The resulting average concentration was 100.4 ng/uL.



<span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Custom 2º PCR (07/08/13) <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">2º PCR was performed using custom primers that were ordered on 07/03/13. A band was present at ~1500 nucleotides - corresponding with the target gene length of 1590 nucleotides. PCR² was then performed on the 2º PCR product.



__<span style="font-family: 'Times New Roman',Times,serif; font-size: 20px;">Week 5 __

<span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Midi-Prep of pNIC-Bsa4 (07/05/13) <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">An overnight culture of pNIC-Bsa4 was prepared on 07/02, spun down and frozen at -20°C on 07/03. Midi-Prep was performed on 07/05, resulting in an average concentration of 38 ng/μL, a 260/280 value of 1.91, and a 260/230 value of 2.85. A sample was sent to sequencing on 07/05 using pLIC-for as the primer.





<span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">PCR Primer Overlap with Q5 Polymerase (07/02/13-07/03/13) <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">An oligo mix was created from primers D4-G5 from primer box B. Primary PCR was performed using the oligo mix. Secondary PCR was performed using primers D4 and G5 as the 'tail primers'.



<span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Tail Primer Design (07/01/13) <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Forward and reverse primers were designed for the DNA sequence coding for Dihydrofolate Reductase-Thymidylate Synthase in L. major.



__<span style="font-family: 'Times New Roman',Times,serif; font-size: 20px;">Week 4 __ <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Restriction Enzyme Digest (06/28/13)

<span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Protein Sonification, Purification, and Characterization (06/26/13 -06/27/13)





<span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Making an SDS-Page Gel (06/25/13) <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Gels were made according to the SDS-Page Gel protocol, with a bottom resolving layer and an upper stacking layer. The first gel that was made had a very wide upper stacking layer with bubbles that created large crevices in the wells. A second gel was made the same day and stored at 4°C for use in the protein characterization lab the next day.

<span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Analyzing DNA Sequence (06/24/13) <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">The BLAST database was used to determine the identity of a plasmid DNA sequence sent to the CMB Core. After determining that the sequence was sufficiently similar to that of pGBR22 it was inserted into the DNA sequence of E. coli in the place where it would be cloned into in wet lab. The newly transformed sequence was run through a virtual enzyme digest. The results were compiled to create a virtual gel detailing how various enzymes would cut the new plasmid (i.e. the number and size of bands created).

__<span style="font-family: 'Times New Roman',Times,serif; font-size: 20px;">Week 3 __

<span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Firefighter Training! (06/19/13)

<span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">PCR Cloning of pmCHERRYin the MCS Site (06/19/13 - 06/20/13)

<span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Large-Scale Protein Expression (06/17/13 - 06/18/13) <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">The FabI plasmid in pNIC-Bsa4 was grown in an overnight culture on 06/17, but was not successful. This was likely due to the old age of the colonies used to start the culture. PfDXR in pNic-BSSA4 from another group was used for the Protein Expression protocol. Cells were grown further, activated with IPTG and centrifuged for harvesting. The pellet with a weight of 2.45g, was suspended in buffer and stored in the -80°C freezer.

<span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">My Second (and More Successful) PCR! Run of pGBR22 (06/18/13)

<span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Week 2 <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Mid-Prep of pmCHERRY (06/11/13)



<span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">The final solution yielded an average concentration of 97.8 ng/uL <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">My First PCR! Run of pGBR22 (06/12/13)

__<span style="font-family: 'Times New Roman',Times,serif; font-size: 20px;">Week 1 __ <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Tony - put in the Weeks in reverse chronological order. <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Also where is your 1st PCR? of pGBR22. <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Those plates look pretty dried out - were they in the incubator for 2 days? - DR. B <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Quantifying DNA Using NanoDrop (06/03/13)



<span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Average concentration of pmCHERRY was 119.05 ng/μL <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Average absorbance at 230nm was .871 <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Average 260/280 ratio value was 1.91 <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Average 260/230 ratio value was 2.73

<span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Transformation Efficiency (06/05/13) <span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 15px; text-align: center;">Images shown in Figures 3-5 were taken on June 10, 2013 - 5 days after being plated. This delay in image capturing accounts for the poor survivorship of the colonies. Colonies were counted the day after plating.







<span style="font-family: 'Times New Roman',Times,serif; font-size: 15px;">Submitting DNA to DNA Sequencing Facility (06/06/13)** <span style="font-family: 'Courier New',Courier,monospace; line-height: 1.5;">NNACNNNTCNGTNNCNCNNNAANCCGTGNNGNNGCNGGCNANANNNNTNNCCTCCNNNNCNANNNACNNNNANNANNCNGCAGNNNNNCTNTGGGTNTATTTAAATNTNNN <span style="font-family: 'Courier New',Courier,monospace; line-height: 1.5;">NNANNTTNNCNNNNN || <span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">DNA Sequence of M13Rev-2, Order Number 91686, from ICMB Core
 * <span style="font-family: 'Courier New',Courier,monospace;">NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN <span style="font-family: 'Courier New',Courier,monospace; line-height: 1.5;">TCTNANNCNNNNNCNCNNNGNNNTGGCAGGNNATTGNNNTNTGNNNNNTGGNCACTGNNNNNNNNNNCGTANGCNTN