Monica+L.+(Springer)

Weeks 14&15

__11/6/14__ Performed a second RE digest in preparation for annealing and transformation. Used BsaI to cut pNIC-Bsa4. Analysis: RE digest appears somewhat successful; a band appeared at SacB gene (about 2kb). However, an additional band in the top section is odd. This may be due to contamination. I continued with annealing and transformation despite this.
 * RE Digest**

__11/6/14__ Created cohesive ends, annealed, and transformed recombinant pNIC-bsa4 into competent DH5a cells.
 * Annealing and Transformation**

Analysis: Some colonies appeared, which should indicate that those colonies were successfully transformed with the pNIC containing lmo1800 and not the SacB gene. The next step will be to create master plates, isolate the DNA, and sequence it to confirm successful cloning.

__11/6/14__ Selected ligands from known inhibitors of other protein tyrosine phosphatases (from different bacteria) as positive controls. Selected compounds with similar chemical properties but no known binding as the negative controls. Ranking and docking was performed by GOLD.
 * Control Ligand Docking**

Analysis: Control screening verified the GOLD docking program by differentiating between positive controls and negative controls. On average, known inhibitors scored better than negative controls, which indicates that GOLD can differentiate between inhibitors and compounds that merely have similar chemical properties. The next step is to screen larger libraries of novel ligands, beginning with the ChemBridge diversity library.

12042014- Fantastic job

Weeks 11,12,&13

__11/6/14__ Performed PCR Cleanup of PCR-squared results to get rid of excess dNTPs, enzymes, failed PCR fragments, etc. and purify the gene. Eluted in Tris HCl. Analysis: PCR Cleanup resulted in a relatively low yield, though workable (30.2 ng/uL). The next step is to prepare and cut pNIC-Bsa4 to prepare for cloning.
 * PCR Cleanup of //lmo1800//**

__11/7/14__ pNIC-Bsa4 was cut by BsaI using 55.83ul of pNIC-plasmid and the reagents were tripled to compensate. More pNIC was used because the yield was low (around 24ng/ul). Results were stored Nanodropped and stored at -20 degrees C. Analysis: RE digest was done to create a space in the vector for the insert. BsaI cut out the SacB gene to allow a space for //lmo1800//. Average yield from 2 trials of Nanodrop was 72.6 ng/ul.
 * RE Digest of pNIC-Bsa4**

__11/17/14__ Used SWISS-MODEL to obtain top hit, which was M. tuberculosis PTP and had 31% identity with Lmptp. Used that PDB structure through ICM to create a homology model for Lmptp. Analysis: The identity was low for this structure compared with Lmptp, and the pairwise comparison showed 26% identity, but this was the highest match we could find. Molprobity was also pretty poor. Will proceed with control ligand docking to check results and if needed, try to come up with a new homology model.
 * Homology Model Creation**

__11/18/14__ Created cohesive ends in the insert and vector. For the insert, 20ul total of BSA, PCR Cleanup, dCTPs, T4 DNA polymerase, DTT, and Nanopure were combined and incubated at 22oC for 30 minutes, then heat inactivated at 75oC for 20 minutes. The same was done to the vector, but at a 50ul total volume and with dGTPs instead of dCTPs. Ratios used: Tube A: 2 vector:4 insert Tube B: 4 vector:6 insert Analysis: Plates lacked colonies; there was no growth. Errors could have resulted from contamination, incorrect ratios, or failed RE digest. Next step is to repeat cloning a second time.
 * Cohesive end generation/Annealing & Transformation Trial 1**

__11/21/14__ Repeated protocol from 11/18/14 but with a Tube C with the ratio 4 vector:8 insert. Analysis: Cloning failed again. Realized that this is most likely due to the fact that too much time had passed between RE digest and cohesive end generation, so it is possible that the plasmid ends reformed in solution over time after being cut by BsaI. Next step will be to repeat RE digest (may have to grow up more pNIC) and try cloning again.
 * CEG/Annealing & Transformation Trial 2**

1162014- Great Job Weeks 8,9,&10

__10/16/14__ Performed trial 3 of primary PCR of //lmo1800// with 1mM oligo mix. Used NEB Recommended thermal settings and adjusted the annealing temperature to around the melting temperature of our oligo mix (derived from DNAWorks results).
 * Primary Overlap PCR #3**

Thermal cycler conditions: 98oC 30 sec. 98oC 7 sec. 62.5oC 20 sec. 72oC 25 sec. 72oC 2 min. 4oC Infinite



Analysis: There was a faint smear in both Tyler and I's PCR wells, indicating successful primary PCR; the oligos were roughly pieced together. We proceeded with secondary PCR to try to create a coherent //lmo1800// gene from the primary PCR products. However, this yield turned out to be insufficient for secondary PCR.

__10/17/14__ Performed trial 1 of secondary PCR using previously ordered forward and reverse primers and 1uL of the template (the product of primary PCR). Used the same thermocycler settings as the primary PCR #3 settings (performed 10/16/14).
 * Secondary PCR #1**

Analysis: Secondary PCR failed; there were no bands visible in lanes 3 and 4, where the PCR mix was run. This could have been due to an annealing temperature that was too high; instead of gauging the annealing temperature to the primer melting point, we used the oligo melting point. In the next trial we will use a lower temperature.

__10/21/14__ Repeated secondary PCR using the same NEB recommended thermal cycler settings and protocol as in trial 1, but changed the annealing temperature to 54.5oC, which is 5oC below the melting point of the primers (~60oC).
 * Secondary PCR #2**



Analysis: Secondary PCR failed again, despite using an annealing temperature 5oC below the primer melting temperatures. This may be due to an insufficient yield from primary PCR (we got a very faint smear only), which may mean there is not enough reaction product to serve as a template in secondary PCR. We will repeat primary PCR with more oligo mix to try to obtain a greater yield.

__10/28/14__ Repeated primary PCR protocol from trial 3 (10/16/14), but used 2uL of oligo mix instead of 1uL. Also used 1uL of Q5 polymerase instead of 0.5uL. Thermal cycler settings were the same.
 * Primary Overlap PCR #4**



Analysis: After repeating primary PCR with extra oligo mix and Q5, we got a much more defined smear. We will proceed to run secondary PCR using the products from this primary PCR.

__10/30/14__ Repeated secondary PCR protocol from trial 2 (10/21/14) with a 54.5oC annealing temperature using the products from primary PCR trial 4 (10/28/14) as the template.
 * Secondary PCR #3**



Analysis: Secondary PCR was successful; a light band appeared at around 900bp (just below the 1kb line), which is the approximate length of the //lmo1800// gene. Oligos were thus successfully pieced together to form //lmo1800//, which can be now used for PCR-squared amplification.

__10/31/14__ Ran PCR-squared to amplify the //lmo1800// from secondary PCR. Made only one PCR tube and used 9uL template from secondary PCR. Used same thermal cycler settings as in secondary PCR #3 but for 30 cycles. Analysis: PCR-squared was successful. A dark band appeared at the ~900bp level, again, the approximate length of our gene; thus, the gene was amplified. We will repeat PCR squared with more PCR tubes to generate more of our gene in preparation for cloning. We will also begin cutting the pNIC-Bsa4 and perhaps will grow up more pNIC since our previous yield was low.
 * PCR-squared **

9232014- Outstanding work Weeks 5,6,&7

__9/29/14__ I designed the tail primers for secondary PCR of the //lmo1800// gene. I used NEB Cutter to define where BsaI would cut the pNIC-Bsa4 plasmid prior to insertion of the gene.
 * Tail Primer Design**



Analysis: These forward and reverse primers will be used during secondary PCR to create a full, cohesive //lmo1800// gene that can be amplified and inserted into the pNIC-Bsa4 plasmid. This will occur once the BsaI RE has cut the gene at the above points (removing the SacB gene).

__10/2/14__ I purified the pNIC-Bsa4 plasmid from the DH5a cell pellet obtained after transformation. This was done by lysing the cells, progressively filtering the products, and finally eluting the sample with TE buffer.
 * Midi-Prep #1**

Analysis: Midi-prep using my cell pellet resulted in a fairly low yield. This may be due to the fact that pNIC-Bsa4 is a low-copy plasmid or that this transformation just happened to result in a lower yield.

__10/3/14__ Repeated midi-prep, but used Tyler's cell pellet instead of mine. Eluted with 500uL TE buffer rather than 1mL (twice as concentrated as last time).
 * Midi-Prep #2**

Analysis: Yield was still low, but plasmids are usable. Considering we eluted the sample with half as much TE, the concentration of the pNIC-Bsa4 was almost the same as in midi-prep #1.

__10/5/14__ I did overlap PCR using 1mM oligo mix (made previously with oligos ordered using DNAWorks output). Thermocycler cycling conditions: 98oC 2 min 98oC 20 sec 58oC 10 sec 72oC 20 sec 72oC 2 min 4oC Infinite Repeat 2-4 for 20 cycles.
 * Primary (Overlap) PCR #1**


 * Forgot to take picture prior to disposing of the gel. Lane 2 was 1kb ladder; all lanes with sample were blank.**

Analysis: There was no smear in the lanes with samples, but the 1kb ladder did appear (problem was not with the gel itself). We made an error in the Q5 dilution (did a 1:2000 dilution of stock rather than 1:2 dilution). Too little Q5 may have resulted in the failed PCR.

__10/7/14__ Repeated thermocycler conditions from overlap PCR #1, but used correct 1:2 dilution of stock Q5 this time. Analysis: PCR failed again; ladder showed up on gel, but no smear/bands. This might be due to the thermocycler conditions. We plan on adjusting the annealing temperature to be closer to the melting temperature of the primers (around 68oC).
 * Primary (Overlap) PCR #2**

09232014-Nice work

Weeks 3&4

__9/12/14__ I selected one colony from the transformed plate and grew it up overnight at 37 degrees in a shaking incubator. I then centrifuged the solution for 15 minutes at 4 degrees C and 6000xg, decanted the liquid, and stored the pellet at -20 degrees C.
 * Bacterial Transformation (days 2-3)**

Analysis: Successful culturing of single colony; solution was cloudy following overnight incubation/shaking (not pictured). Again, cells likely were transformed by the pNIC-Bsa4 because the KAN should have killed off any unsuccessfully transformed bacteria. This could be confirmed with protein characterization.

__9/14/14__ I used the enzymes EcoRI, PvuII, and a combination of the two to cut the pGBR22 plasmid in preparation to receive the gene to be cloned. This was done by incubating a mixture of buffer, enzyme, water, and plasmid at 37 degrees C and then heat shocking to kill the enzyme. Results were run through agarose gel to confirm that enzyme was cut in the correct places.
 * Restriction Enzyme Digest**

Analysis: RE digest was successful. The gel revealed that the plasmid was cut in the correct places (matched previous virtual gel in location and number of bands). The EcoRI, a 1-cutter enzyme, resulted in 1 band, the PvuII (2-cutter) resulted in 2, and the combined usage of the two resulted in 3 bands, as expected. Some contamination occurred (unrelated bands shown on the gel). Plasmids would now be ready for gene insertion.

__9/15/14__ I created a Master Mix using DNTPs, M13F and M13R primer, buffer, and water. I added this to diluted template DNA with the Taq polymerase and ran the PCR. Results were run through agarose gel.
 * My First** **PCR**

Analysis: PCR was successful! Clear bands appeared at the correct distance for each concentration. As expected, higher concentrations of template DNA resulted in thicker bands. The next step would be bacterial transformation using the amplified gene.

982014- Good Job

Weeks 1 & 2

__9/2/14__ We began the transformation by Nanodropping unverified pNIC-Bsa4 plasmid. The 260/280 and 260/230 values were around 1.8 and 2.1, respectively, indicating a relatively pure sample. We performed the transformation using 50ng of the plasmid and 25ul E. coli bacteria with SOC media. We placed 10ul onto one LB agar plate and 50ul onto another and allowed overnight incubation at 37 degrees Celsius.
 * Bacterial Transformation (day 1)**



Analysis: Successful colony growth, with the 50ul plate showing more colonies than the 10ul plate, as expected. Colonies shown likely were transformed by the pNIC-Bsa4 because the KAN should have killed off any unsuccessfully transformed bacteria. However, this has not been confirmed yet.