Jennifer+R.

__gene sequence__ ATGACGACATCTGCTCTATCTCATCCTTCCCTATTGCCACTGGATGGGGGTATTAACTTCCGTGATCTCG GTGGTAACGTCGTTGCAGATGGCCGGCGTATTAAGCGCGGGTTATTGTTTCGTTCAGGATCACTTGATCG TTTGAGCACCAAAGATTGTGCTGTTCTTAGCAGCGGCTCTGTCGCTCAAATCCTTGATTACCGCGATGCG GATGAAGTTCAGGCTAAGCCCGATATTGTGTGGCAAGGTGCCAGTTATCACAATATTCCAGCCAACCCAC CCAGCAGTGAAGTTAACGCCAATCTGGAAAAACTGACTAACGAGACGTTAGCAACATTTGATGCCAGAGC TTTCATGTTAGAGCTGTACCGTCGCCTGCCATTTAATAATCAAGCTTATAAACAGTTAGTCAGTTTGCTA CAAAATAGTGCCTCACCAGAGCATGCCGCTGCTGGCGTGGTACAGCATTGTGCAGTCGGGAAAGACCGTA CTGGAGTGGGTTCGGCTTTGGTGTTGTTTGCTTTAGGTGCTGATGAATCGACGGTATTGGAAGATTACCT GTTGACTGAAACAACACTGGCACCTTTCCGTGAACACATGCTGGCGGAATTGGCACTAAAACTAAATTCT CAGGCGCTAGGACAGTTTGCATTTGTGTTATCGGCTCGAGAAGAGTTTATTCAAACCGCACTGCGATGTA TTCAAGAGCGTTACGGGAGCAGCGAGCAATGGCTACAGCAGGAATTTGGTCTTGGCAGAATTGAGCGCGA AAAGTTGCAGTCATATTTCCTGGAGTAA

__December 6, 2012__ 10 best docking poses with scores screened against CBkin_UT library
 * **

__December 5, 2012__ Compound enzyme5154121 was used.
 * **


 * Absorbance at 410nm**


 * Run1 || Reading 1 || Reading 2 || Avg || Avg-enzyme control || STDV ||
 * A1 || 0 || 0 || 0 || 0 || 0 ||
 * B1 || 0 || 0 || 0 || 0 || 0 ||
 * C1 || 0.036 || 0.033 || 0.0345 || 0.0345 || 0.002121 ||
 * D1 || 0.033 || 0.033 || 0.033 || -0.0015 || 0 ||
 * E1 || 0.026 || 0.027 || 0.0265 || -0.0065 || 0.000707 ||
 * F1 || 0.072 || 0.075 || 0.0735 || 0.047 || 0.002121 ||
 * G1 || 0.052 || 0.047 || 0.0495 || -0.024 || 0.003536 ||
 * H1 || 0.063 || 0.064 || 0.0635 || 0.014 || 0.000707 ||
 * I1 || 0.002 || 0.003 || 0.0025 || -0.061 || 0.000707 ||



Result: I found the reason why the absorbance is way too low. I stored the concentration buffer #1 in the -20 without glycerol.
 * Run2 || Reading 1 || Reading 2 || Avg || Avg-enzyme control || STDV ||
 * A1 || 0 || 0 || 0 || 0 || 0 ||
 * B1 || 0 || 0 || 0 || 0 || 0 ||
 * C1 || 0 || 0 || 0 || 0 || 0 ||
 * D1 || 0 || 0 || 0 || 0 || 0 ||
 * E1 || 0 || 0 || 0 || 0 || 0 ||
 * F1 || 0 || 0 || 0 || 0 || 0 ||
 * G1 || 0 || 0 || 0 || 0 || 0 ||
 * H1 || 0 || 0 || 0 || 0 || 0 ||
 * I1 || 0 || 0 || 0 || 0 || 0 ||

__December 4, 2012__
 * **

Run 1 || Absorbance Run2 || Absorbance Avg || Absorbance Avg-A || STDV ||
 * Absorbance at 410nm**
 * || Absorbane
 * A || 0.054 || 0.056 || 0.055 || 0 || 0.001414 ||
 * B || 0.056 || 0.058 || 0.057 || 0.002 || 0.001414 ||
 * C || 0.128 || 0.125 || 0.1265 || 0.0715 || 0.002121 ||
 * D || 0.082 || 0.081 || 0.0815 || 0.0265 || 0.000707 ||
 * E || 0.111 || 0.109 || 0.11 || 0.055 || 0.001414 ||
 * F || 0.134 || 0.131 || 0.1325 || 0.0775 || 0.002121 ||
 * G || 0.204 || 0.201 || 0.2025 || 0.1475 || 0.002121 ||
 * H || 0.153 || 0.153 || 0.153 || 0.098 || 0 ||

__December 3, 2012__
 * **


 * Gel image**

Lane 1: marker Lane 2: sample 0- cell lysate before induction Lane 3: sample 1- cell lysate after induction Lane 4: sample 2- soluble fraction Lane 5: sample 3- flow through Lane 6: sample 4- wash Lane 7: sample 5- elution 1 Lane 8: sample 6- elution 2

Lane 1: marker Lane 2: sample 0- cell lysate before induction Lane 3: sample 1- cell lysate after induction Lane 4: sample 2- soluble fraction Lane 5: sample 3- flow through Lane 6: sample 4- wash Lane 7: sample 5- elution 1 Lane 8: sample 6- elution 2
 * Dried Gel image**



The samples was concentrated and measured A280nm on Nanodrop with using storage buffer as blank.
 * **

The result:

Concentrated Elution Buffer 1

Calculation based on Googledocs.
 * || Volume of LB Used || A || E [1/Molar 1/cm] || b || c [Molar] || uM || MW [g/mol] || g/L = mg/ml || ng/ul || <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">Total mg in Xml || <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">Xml - elution volume || <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">260/280 ||
 * <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">Concentrated Elution 1 || <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">500mL || <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">1.209 || <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">53290 || <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">1 || <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">2.27E-05 || <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">22.69 || <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">39528.5 || <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">0.90 || <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">897 || <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">4.48 || <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">5 || <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">0.95 ||

__December 1, 2012__ After the centrifuging sample 0 and 1, only the pellets were kept and water and 6x gel loading buffer was added. 6x gel loading buffer was added to all the other samples. The gel was placed in the mini-Protean tank and the samples were run with marker. After 35min passed, the gel was taken out and placed into the Petri dish with nanopure water. Gel was washed with the nanopure water for three times. Then, the Recycled Imperial Stain was poured into the dish and placed on an orbital shaker for over an hour. Afterwards, the gel was distained with nanopure water twice. Tissue was folded and placed in the dish to soak up excess stain and wash the gel overnight to remove the background staning.
 * <Protein Characterization>**

The samples were measured A280nm on Nanodrop with using elution buffer as blank.
 * <Measure OD of Elution #1 and #2 samples>**

The result:

Elution Buffer 1

Elution Buffer 2

Calculation based on Googledocs.
 * || Volume of LB Used || A || E [1/Molar 1/cm] || b || c [Molar] || uM || MW [g/mol] || g/L = mg/ml || ng/ul || Total mg in Xml || Xml - elution volume || 260/280 ||
 * Elution 1 || 500mL || 0.32 || 53290 || 1 || 6.00E-06 || 6.00 || 39528.5 || 0.24 || 237 || 1.19 || 5 || 0.71 ||
 * Elution 2 || 500mL || 0.05 || 53290 || 1 || 9.38E-07 || 0.94 || 39528.5 || 0.04 || 37 || 0.19 || 5 || -2.63 ||

__November 30, 2012__ 1mL of Ni-NTA was added into the sample tube and incubated on the ice for 45min. The, it was transferred in to the chromatography column. It was important that in the column, the level of buffer should keep above the top of the resin to prevent affinity matrix gets dry. [Flow through step ] After the material in the column has settled, the sample #3 was collected. [Wash step] to remove proteins that are only loosely bound to resin. Ni-NTA resin was wash with wash buffer and sample #4 was collected. [Elution step] the tagged protein can be released from Ni-NTA resin by using a buffer with a high concentration of imidazole. The bound protein was eluted with Elution buffer. Eluted protein was keep on ice. The buffer was collected on the ice and sample #5 was collected. More elution buffer was added and sample #6 was collected. All the sample collected was stored in the 4degree Celsius with other samples collected before to run on SDS-PAGE Gel.
 * <Protein Purification>**

Day 5. The sample in the conical tube was transferred into the centrifuge tube. Then, it was spin down in the big centrifuge. The 50uL of sample #2 was collected and stored in the 4degree Celsius with other samples collected before. The supernatant was transferred into the conical tube and pH was checked. It had pH of 7.9. Then, the syringe filter was processed with 0.45um PES. The protein purification protocol was performed right after.
 * <Protein Expression>**

The top 10 ligands with polar contacts were examined in the pymol and pasted into the lab notebook.
 * <Virtual Screen YEPTP>**

__November 29, 2012__

Day 4. The tube containing pellet was thaw on the ice. After that, the sonication was proceeded while surrounding the tube in ice within a glass beaker. It was load onto platform so that sonicator tip does not touch tube. The Output control was set at 5.5 and the four steps were processed. Then, the spin down step was performed and sample#2 was saved.
 * <Protein Expression>**

Finished 2nd GOLD run and obtained top 10% ligand.
 * <Virtual Screen YEPTP>**

__November 27, 2012__ 1st GOLD run submitted before Thanksgiving break and obtained top 10% ligand. The 2nd GOLD run was submitted.
 * <Virtual Screen YEPTP>**

112612 - Good. Dr B

__November 20, 2012__

__Day 3.__
 * <Protein Expression>**

The fresh, pre-made LB was warmed up in the incubator. The sample stored in the 4degree Celsius freezer was warmed to room temperature and 10mL of the sample was added into the 500mL LB in 1L flask. The OD at 600nm was measured and recorded. Culture was keep added within 5mL increments until OD600 is around 0.1 Then, the 500uL Kan was added and stored into the incubator. OD600 was checked every 30minutes until 0.5. However, I stopped at the 0.332 since time was running out. 500uL of Sample #0 was collected, then in the flask 250uL of 1M IPTC was added. The flask was stored in the incubator and sample was stored in the 4degree Celsius freezer. After 2.5hr pasesd, the flask was takend out and 500uL of sample #1 was collected after induction. The 500mL is separated into two bottles and put into 37degree Celsius big shaking incubator in the 1st floor for 20min. The pellet are stored in the 4 degree Celsius.

Result:
 * Time || OD at 600nm ||
 * 0min || 0.11 ||
 * 30min || 0.138 ||
 * 1hr || 0.128 ||
 * 1.5hr || 0.157 ||
 * 2hr || 0.332 ||




 * <Virtual Screen YEPTP>**
 * Resolution || 1.8 Å ||
 * Clashscore, all atoms || 5.3 ||
 * Poor rotamers || 0.21% ||
 * Ramachandran outliers || 0% ||
 * Ramachandran favored || 97.14% ||
 * Cβ deviations >0.25Å || 0 ||
 * MolProbity score^ || 1.44 ||
 * Residues with bad bonds || 0% ||
 * Residues with bad angles || 0% ||
 * Error in Active Site ||  ||

1chain of YEPTP which has active site
 * <span style="display: block; font-family: Times,serif; font-size: 12pt; text-align: center;">All-Atom

<span style="display: block; font-family: Times,serif; font-size: 12pt; text-align: center;">Contacts || <span style="font-family: Times,serif; font-size: 12pt;">Clashscore, all atoms: || <span style="font-family: Times,serif; font-size: 12pt;">5.76 || <span style="font-family: Times,serif; font-size: 12pt;">91st percentile* (N=1784, all resolutions) ||
 * ^  |||||| <span style="font-family: Times,serif; font-size: 12pt;">Clashscore is the number of serious steric overlaps (> 0.4 Å) per 1000 atoms. ||
 * <span style="display: block; font-family: Times,serif; font-size: 12pt; text-align: center;">Protein

<span style="display: block; font-family: Times,serif; font-size: 12pt; text-align: center;">Geometry || <span style="font-family: Times,serif; font-size: 12pt;">Poor rotamers || <span style="font-family: Times,serif; font-size: 12pt;">0.42% || <span style="font-family: Times,serif; font-size: 12pt;">Goal: <1% ||
 * ^  || <span style="font-family: Times,serif; font-size: 12pt;">Ramachandran outliers || <span style="font-family: Times,serif; font-size: 12pt;">0.00% || <span style="font-family: Times,serif; font-size: 12pt;">Goal: <0.2% ||
 * ^  || <span style="font-family: Times,serif; font-size: 12pt;">Ramachandran favored || <span style="font-family: Times,serif; font-size: 12pt;">96.79% || <span style="font-family: Times,serif; font-size: 12pt;">Goal: >98% ||
 * ^  || <span style="font-family: Times,serif; font-size: 12pt;">Cβ deviations >0.25Å || <span style="font-family: Times,serif; font-size: 12pt;">0 || <span style="font-family: Times,serif; font-size: 12pt;">Goal: 0 ||
 * ^  || <span style="font-family: Times,serif; font-size: 12pt;">MolProbity score <span style="font-family: Times,serif; font-size: 10pt;">^ || <span style="font-family: Times,serif; font-size: 12pt;">1.51 || <span style="font-family: Times,serif; font-size: 12pt;">95th percentile* (N=27675, 0Å - 99Å) ||
 * ^  || <span style="font-family: Times,serif; font-size: 12pt;">Residues with bad bonds: || <span style="font-family: Times,serif; font-size: 12pt;">0.00% || <span style="font-family: Times,serif; font-size: 12pt;">Goal: 0% ||
 * ^  || <span style="font-family: Times,serif; font-size: 12pt;">Residues with bad angles: || <span style="font-family: Times,serif; font-size: 12pt;">0.00% || <span style="font-family: Times,serif; font-size: 12pt;">Goal: <0.1% ||

__November 19, 2012__

Proten Tyrosine Phosphatase homology model was re-submitted. Result:
 * <Homology Model>**

__Day 2.__ LB media from the refrigerator was warmed in the incubator. In the two 500mL flasks, 100mL of LB media were inoculated. Two separate single colonies of FtHap cells pre-made from overnight transformation were added into the flask by using sterile pipette tip. The antibiotic was added to final concentration of 50 ug/ml Kan. Therefore, total 100uL of Kan was added to each flask since they have 100mL volume of LB media. Two flasks were placed into the shaking incubator at 37 degree Celsius for overnight. ~12 hours, the flasks were stored in the 4 degree Celsius until next day.
 * <Protein Expression>**

__November 18, 2012__ Jennifer - include some 'data' or graphs here. Dr. B 11/19/12

Proten Tyrosine Phosphatase homology model had failed. The result is saying, - Start BLAST for highly similar template structure identification - No suitable templates found! - Run HHSearch to detect remotely related template structures - Unfortunately, we could not identify useful template structures
 * <Homology Model>**

__November 16, 2012__

__Day 3.__
 * <Protein Expression>**

Could not proceed to the day 3 since the flask in the incubator might be dried out. Therefore, I'll restart the process. LB media was running out, so I made LB 2.0L of LB media following by making LB media protocol.

Proten Tyrosine Phosphatase sequence was put into the swiss-model website to create a homology model.
 * <Homology Model>**

__November 15, 2012__


 * <Protein Expression>**

__Day 2.__

LB media from the refrigerator was warmed in the incubator. In the two 500mL flasks, 100mL of LB media were inoculated. Two separate single colonies of FtHap cells pre-made from overnight transformation were added into the flask by using sterile pipette tip. The antibiotic was added to final concentration of 50 ug/ml Kan. Therefore, total 100uL of Kan was added to each flask since they have 100mL volume of LB media. Two flasks were placed into the shaking incubator at 37 degree Celsius for overnight. ~12 hours, the flasks were stored in the 4 degree Celsius until next day

__November 9, 2012__


 * <pNIC-Bsa4 cloning>**

__Part 4. Making Master Plate__

There were any colonies shown on the both plates. Those two plates were thrown away into the bio-hazard bin. Therefore, I couldn't go onto the next step. Since cloning process has failed, I might make MtPSTP as surrogate next week.

__November 7, 2012__


 * <pNIC-Bsa4 cloning>**

__Part 1. Preparation of pNIC-Bsa4 as Accepting Vector Gel Check__

__Result:__ The gel image of pNIC-Bsa4 as Accepting Vector Lane 1: DNA 1kb ladder Lane 2: Ivy's pNIC-Bsa4 Lane 3: My pNIC-Bsa4. It had two distinct bands which shows the pNIC-Bsa4 was cut into two fragments by ECO311.

__Part 2. Cohesive End Generation on PCR Inserts and Accepting Vector__

The sample solutions were made and placed in the water bath for 30min at 22 degree Celsius. And then, they were heat inactivated for 20min at 75 degree Celsius. __Result:__ The nanodrop graph of pNIC-Bsa4 as Accepting Vector

__Part 3. Annealing and Transformation__

This part was done immeditely after Cohesive Ends generation of accepting vector and PCR insert. Tube A was the mixture of 2uL of T4-treated Accepting Vector with 4uL of each T4-treated Insert. Tube B was my trial with the mixture of 12uL of T4-treated Accepting Vector and 6uL of T4-treated Insert. They were stored at the room temperature for 10 minutes, and then transferred to ice. 25uL of DH5alpha Competent cells were added into the ligation mixture in the tubes, and incubated on ice for 30minutes. Then, they were heat shocked in the water bath for exactly 30 seconds in 42 degree Celsius, placed back on the ice bucket, and added 100uL of pre-warmed SOC. These two tubes were shaked in the 37 degree Celsius shaker for an hour. After an hour passed, the transformation mixture of two tubes were spread to plate of LB-Agar, Kanamycin, and sucrose with colirollers. They were then incubated upside down overnight at 37 degree Celsius for a day.

__November 5, 2012__

__Part 1. Preparation of pNIC-Bsa4 as Accepting Vector__
 * <pNIC-Bsa4 cloning>**

The sample was made of pNIC-Bsa4 plasmid, Fermentas Buffer, 100X BSA, ECO311 from Fermentas, and nanopure water. The volume of pNIC-Bsa4 was calculated but the amount was over 25uL which is the final volume. So, all the reagents used were scaled up with 1.4. Thus, the final volume become 35uL instead of 25uL. This sample was placed in the water bath of 37degree Celsius for 2 to 3 hours. After the incubation, the sample was PCR cleaned up and then nanodropped to find the final concentratoin.

__Results:__ The nanodrop graph results after PCR cleanup of pNIC-Bsa4

__October 26, 2012__


 * <PCR Primer Overlap>**

__Part 4. PCR squared reaction__ There will be great lose in the PCR cleanup procedure after the PCR squared reaction, therefore the PCR squared reaction is needed to make a lot of PCR product. I made three samples of PCR squared reaction, Yellow tube from the sample of second lane in secondary PCR, Pink tube from the sample of third lane in secondary PCR, and one tube from the Ivy's secondary PCR sample. The total volume of 200uL of sample was made, and divided into 4 tubes and run on the PCR machine.
 * The pink tube from the sample of third lane in secondary PCR was the one had distinct bands on the gel.

__Part 5. PCR cleanup__ The red box of PCR cleanup kit was used. The 4 sample tubes were combined into one sample. After each solution was added, the sample was centrifuged with maximum speed, then the eluate was decanted. Only the eluate from the last step which is my DNA sample to clone was conserved.

__Part 6. Nanodrop__ The eluate was nanodropped. First, the sterile water was used to calibrate the spectrophotometer. Second, the elution buffer was used to blank. Lastly, the sample was used to measure.

__Results:__ The nanodrop graph results after PCR squared and cleanup <span style="font-family: 'Calibri','sans-serif'; font-size: 14.6667px;">Result from second lane sample of secondary PCR. The graph looks like there is a contamination.

Result from third lane sample of secondary PCR. The graph looks good with high concentration.

Result from Ivy’s sample. Graph looks good but the concentration is lower than my sample.

__October 19, 2012__ 102112- Jennifer - ok. If you can't get secondary to work, then use soem of Ivy's sample to make your PCR squared - Dr. B

Part 3. Secondary PCR Re-run the secondary PCR with 100bp ladder
 * <PCR Primer Overlap>**
 * Result:** Gel image of secondary PCR



Finally bands appeared on the gel. Lane 2. 100bp ladder Lane 3. secondary PCR sample Lane 4. secondary PCR sample Lane 3 or 4 should have distinct band which corresponds to the size of the YEPEP. However, it wasn't shown on the gel for lane 3. But there was bands appeared on lane 4. Lane 6 through 10 is Stephanie's and Aldo's who shared same gel with me.


 * <Virtual Screen Refresher>**
 * Result:**

__October 17, 2012__ Part 3. Primary and Secondary PCR Re-run the primary and secondary PCR with 100bp ladder
 * <PCR Primer Overlap>**


 * Result:** Gel image of primary and secondary PCR

1st trial. Something went wrong. Even after three hours, the samples on the gel didn't move at all. The voltage was keep lowering to 70v. Tried different trays and lid and seemed like working for five minutes, but after that voltage lowered to 70. So of course, couldn't see any bands on the gel image. Guessing the gel was a problem.

Trial 2. Therefore, gel was remade and samples were loaded.

Finally, could see the smear on the lane 4 which is primary PCR. It's hard to see but when you invert it, then you can see the smear. However, still can't see the secondary PCR. The gel was torn at the bottom before I capture the image. Still don't know why the distinct bands are not showing when secondary PCR sample was run.

__October 15, 2012__ Part 3. Secondary PCR Re-run the secondary PCR with 100bp ladder The result was bad. Only the DNA ladder(100bp) was shown in the lane2 from the right. From the right side, Lane 2. 100bp ladder Lane 4. secondary PCR at melting temperature of 500 degree Celsius Lane 6. secondary PCR at melting temperature of 60 degree Celsius Lane 2 or 4 should have distinct band which corresponds to the size of the YEPEP. However, it wasn't shown on the gel. Actually, could not see any band for lane 4 and 6. Lane 10 is Stephanie's who worked with me. She got good primary sample result as it looks like smear. She used the melting temperature of 58 as in the protocol, and got a result. Therefore, I could conclude that either my oligo mix was contaminated since the well box wasn't stored well or my personal mistake that I didn't catch. I might use Stephanie's primary sample to do secondary PCR since this is my third trial and still couldn't get the result.
 * <PCR Primer Overlap>**
 * Result:** Gel image of secondary PCR

__October 11, 2012__ 101612 - Jennifer, ok crop your gem images better to get rid of the black space. -- Dr. B

Part 2. Primary PCR Re-run the primary PCR with 100bp ladder From the left. Lane 2. 100 bp ladder Lane 3. primary PCR at melting temperature of 50 degree Celsius Lane 4. primary PCR at melting temperature of 60 degree Celsius Primary PCR should look like smear. But on my gel, there is a band-like image on the lane 3 and 4 from the left. Not sure what it is exactly. The result was not good. Lane2 of 100bp ladder looks good though.
 * <PCR Primer Overlap>**
 * Result:** Gel image of primary PCR

__October 8, 2012__ __100912 - the 100bp ladder looks degraded - did you use one that had been sitting out? Or perhaps the whole gel is bad. ?? - Dr B__ Part 3. Secondary PCR Re-run the primary and secondary PCR with 100bp ladder The result was bad. Only the DNA ladder(100bp) was shown in the lane1. Lane 1. 100bp ladder Lane 2. primary PCR sample Lane 3: secondary PCR sample Lane 2 was supposed to look like a smear. Lane 3 should have distinct band which corresponds to the size of the YEPEP. However, it wasn't shown on the gel. Therefore, I couldn't proceed to PCR squared reaction and have to re-do it.
 * <PCR Primer Overlap>**
 * Result:** Gel image of primary and secondaryPCR

__October 8, 2012__ Part 2. Primary PCR Re-run the primary PCR with 100bp ladder From the left. Lane 2. 100 bp ladder Lane 3. primary PCR Primary PCR should look like smear. But on my gel, there is a band-like image on the lane 3 from the lef. Not sure what it is exactly. Lane2 of 100bp ladder looks good though.
 * <PCR Primer Overlap>**
 * Result:** Gel image of primary PCR

__October 4, 2012__ __100912 - Jennifer - ok. keep trying. Crop your gels better so taht only the gel is visible and not all of the black space. - Dr. B__ Part 3. Secondary PCR YEPTP forward and reverse primer finally came. First, forward and reverse primers were diluted to 100uM of stock solution. Second, These 100uM of stock solution was diluted into 20uM of 100uL working dilution. After the secondary primer solution was made, it was run into the PRC machine and then run on the gel with primary primer solution made last time.
 * <PCR Primer Overlap>**


 * Result:** Gel image of PCR Primer Overlap



The result was bad. Only the DNA ladder(100bp) was shown in the lane1. Lane 2: primary PCR sample Lane 3: secondary PCR sample Lane 2 was supposed to look like a smear. Lane 3 should have distinct band which corresponds to the size of the YEPEP. However, it wasn't shown on the gel. Therefore, I couldn't proceed to PCR squared reaction and have to re-do it. There are two biggest possibility of this poor result that I can think. First, the reagents used to make a sample was contaminated. Dr. B told me that KOD reagents might be contaminated since other students also didn't get the result. Second, the melting temperature can be changed to get better result. Maybe lower or higher than 58degree Celsius. Next time, I'll use new KOD reagents in the box and change the melting temperature to get better result.

__Ocboter 2, 2012__
 * <Pymol Refresher>**

__September 28, 2012__ 093012 - Jennifer. Ok good. Show your sequences form Primer design (don't link to a Word document). -- Dr. B Part 1. Preparing oligo mix Primers from the well plate for Yersinia //enterocolitica//(YE) from A1 through B8 since my target was protein-tyrosine-phosphatase(PTP) were mixed together to make oligo mix.
 * <PCR Primer Overlap>**

Part 2. Primary PCR First, I was planning to use Q5 polymerase. But there was only a little amount left, KOD polymerase was used instead of Q5. After the sample was run in PCR machine, it was stored in the freezer for secondary PCR later since the reverse and forward primers of YEPTP hasn't arrived. Also it was not run on the gel since I want to put both primary and secondary PCR samples on the same gel.

__September 27, 2012__

Organism: Yersinia //enterocolitica// Target: protein-tyrosine-phosphatase CDS(coding DNA sequence) was inserted into the pNIC-Bsa4, which is an accepting vector, where cut by the BsaI. Result:
 * <PCR Primer Design for PNIC-Bsa4 Cloning>**

Final Result: TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGCAC CATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGAGAACCTGTACTTCCAATCCATGACCACCTCTGCACTCTCTCACCCATCT CTGCTCCCGCTGGACGGTGGTATCAACTTCCGTGATCTGGGTGGTAACGTTGTTGCGGACGGTCGTCGTATTAAACGTGGTCTGCTGT TCCGCAGCGGTAGCCTGGACCGTCTGTCTACCAAAGACTGCGCTGTTCTCTCTAGCGGTTCTGTTGCGCAGATCCTGGACTACCGTG ACGCGGACGAAGTTCAGGCGAAACCGGACATCGTTTGGCAGGGTGCGTCTTACCACAACATCCCGGCGAACCCGCCGTCTTCTGAAG TTAACGCGAACCTGGAAAAACTCACCAACGAAACTCTGGCCACCTTCGACGCTCGTGCGTTCATGCTGGAACTGTACCGTCGTCTGCC GTTCAACAACCAGGCGTACAAACAGCTGGTTTCTCTCCTGCAGAACTCTGCGTCTCCGGAACACGCGGCTGCGGGTGTTGTTCAGCA CTGCGCGGTTGGTAAAGACCGTACCGGTGTTGGTTCTGCGCTCGTCCTGTTTGCCCTCGGTGCCGACGAATCTACCGTTCTGGAAGA CTACCTGCTGACTGAGACCACCCTGGCGCCTTTTCGTGAGCACATGCTCGCTGAACTGGCTCTGAAACTGAACTCTCAGGCGCTGGG TCAGTTCGCCTTCGTTCTGTCTGCGCGTGAAGAATTCATCCAAACGGCCCTGCGTTGCATCCAGGAACGCTACGGTTCTAGCGAACAG TGGCTGCAACAGGAATTCGGTCTGGGTCGTATCGAACGTGAAAAGCTCCAATCTTATTTCCTCGAATAACAGTAAAGGTGGATACGGATC CGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAA AGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCT GAAAGGAGGAACTATATCCGGATTGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGC GTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCG TCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTT CACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACT GGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTA ACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTT TATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCA GGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTA TCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAG TGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATC ACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGA ATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTT CCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCA GTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATAC AATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGC CTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATC CCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTG CTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTC AGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCG CTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGG CGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGA GCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGG GAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCA GGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCC TGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCG AGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCAC TCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACAC CCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGC ATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATG TCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTT TTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATA CGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCA CTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAAT GGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTT GCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCA ACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACC AGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCT CGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGC CCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACA TTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGG CGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAG AGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCT TCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTG ATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCC TTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGG CCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTG TTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCG GATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCG ACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGT GGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCA CTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACA TCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATT CGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGC CGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGC GCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTG ATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAAT

__September 21, 2012__ Looks good Jennifer - Dr. B

<Nano drop graph of the DNA sample from transformed bacterial cells(pNI-Bsa4) after the Midi prep>
 * <Midi Prep>**


 * <PCR protocol>**
 * Part 2. PCR and Gel**

Result:Gel
 * pLIC sequencing vectors of pNIC-Bsa4**



Only marker was shown. I'll redo it.

__September 20, 2012__

Master mix of pNIC-Bsa4, Forward and Reverse Primers, dNT mix, and ThermoPol Buffer NEB were prepared. In the tubes, equal portion of master mix was added and DNA template and DDW were added. These prepared tubes were stored in the freeze for next steps.
 * <PCR protocol>**
 * Part 1. pLIC sequencing vectors of pNIC-Bsa4**
 * - Preparation of Master mix**

Jennifer - ok good. You can put your figure legend below the gel image. Also, is the PCR band the right size? Does it match the expected size of the gene you are amplifying? - DR. B 091812

__September 15, 2012__

In the tubes in the freeze, diluted Taq DNA polymerase NEB was added and then placed into the PCR machine. PCR was preheated and then thermal program was run. The program took an hour and thirty five minutes, so while waiting, the Agarose Gel was made. Since the rubber of the tray was misplaced, the gel was leaked. So, I had to made another gel. On the gel, samples were loaded and gel was run for about 40 min. The gel was viewed and labeled.
 * <PCR protocol>**
 * Part 2. PCR and Gel**

Results: 1st PCR gel of pGBR 22 with M13 forward and reverse primer. Gel image viewed with UV. The Lane 1 is marker. Lane 2 is Sample A with 1:1000 ratio of Template and 1ng of DNA template. Lane 3 is Sample B with 1:1000 ratio of Template and 10ng of DNA template. Lane 4 is Sample C with 1:100 ratio of Template and 10ng of DNA template. Lane 5 is Sample D no DNA control. There was no band in Lane 3 and 4. The lane 2 and 5 had one thick band.

__September 12, 2012__

Master mix of pGBR22, Forward and Reverse Primers, dNTP mix, and ThermoPol Buffer NEB were prepared. In the tubes, equal portion of master mix was added and DNA template and DDW were added. These prepared tubes were stored in the freeze for next steps.
 * <PCR protocol>**
 * Part 1. Preparation of Master mix**

__September 11, 2012__

It was performed in the late afternoon. On the control plate, there were hundreds of colonies(yellow circular) presented. On the no control plate, there were two bigger size than the colonies on the control plate presented. These were considered as contamination. Since there should be no colonies appeared in no control plate. After adding Kanamycin into the Erlenmeyer flask containing 160mL LB and colony, they were stored in the shaker to let colonies grow up overnight.
 * <Transformation of competent cells for plasmid prep of pNIC-Bsa4>**
 * Day 2.**

Jennifer, put these in reverse order -- Thanks, DR. B 091012

__September 10, 2012__

The two Agar plates were in the incubator over the weekend. So they were moved from 37degree Celcius incubator to 4degree Celcius freeze for the Day 2 experiment tomorrow.
 * <Transformation of competent cells for plasmid prep of pNIC-Bsa4>**

__September 7, 2012__

Worked on the analyzing DNA sequence in the computer lab.
 * <Analyzing DNA sequence>**

Two agar plates were prepared and stored in incubator. One for no DNA control and the oher one for DNA contorl of E.Coli DH5alpha bacteria.
 * <Transformation of competent cells for plasmid prep of pNIC-Bsa4>**
 * Day 1.**

__September 6, 2012__

Practiced dilution of primers with ordered one. pLIC forward was used. Did calculation for the amount of TE and stock solution. Final product of Stock solutions and __#|Working__ Dilutions were stored in the __#|freezer__.
 * <Primer Dilution>**

This lab was performed with the __#|working__ dilution made in the //Primer Dilution// experiment to determine gene present in pNIC-Bsa4. By using m1v1=m2v2, the amount of template, primer, and water were calculated. For the reverse, I used Shane's. The DNA sequencing request form was submitted online and the final solution was taken over to MBB and stored in the fridge.
 * <DNA sequencing facility for pNIC-Bsa4 vector>**

__August 31, 2012__

Target (protein/gene name): 1-deoxy-D-xylulose 5-phosphate reductoisomerase Organism: //Francisella tularensis subsp. tularensis SCHU S4//
 * <PCR Primer Design for Primer Overlap Assembly PCR>**

//Today I worked on primer overlap assembly PCR with my target DXR of organism Francisella tularensis.// //Next week, I'm going to work on// __ProtocolPCR_PrimerOverlap__ <span style="display: block; height: 1px; left: -40px; overflow: hidden; position: absolute; top: 13107px; width: 1px;">