Anh+P.+(Springer)

[[image:ttp453_control ligands.png width="967" height="343" caption="Table 1. Control ligands table: positive and negative controls."]]
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[[image:ttp453_top 30.png width="817" height="567" caption="Table 2. Top 30 from Virtual Screening "]]
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__**Analysis:**__ In the first part, validation of the accuracy of GOLD was done with a library of positive and negative controls. GOLD was used to run the library and a list of best rankings was generated for the poses. Positive controls resulted in higher scores than negative controls as expected. PyMOL was used to analyze the docking of GOLD, and it showed that GOLD docking is accurate because the docking into the active site was really well. The scores range from 38 to 85. In the second part, GOLD was used to run the Chembridge library of 306 ligands. The scores range from 56 to 71 and pyMOL was also used to analyze the top 5 ligands. The next step is to order the top ligands for inhibition assay.

**11/21 -** DNA Sequencing of cloned MtPtpA in pNIC-Bsa4
Sample ATP1_mtptpafor >ATP1-mtptpafor 968 968 0 0.05 NNNNNNNNNNNNNNNNNNNNNNNNNNNANNNNNNNNNNNNNNCTNNNNNTNTGNCAGNNCNNGTNGNNNNNGNATNCTNNGNCNTGTNNGCNNCNCNNNANNNNNNCNATCNNNNNNCNNNNNNNNNC NGNNTNNTTNNNNNNNNNCNNNNNTNNNNNTNCCNNNNNNNNNNNNNNNNNGNNNNNNNNCTGNGNNNNCNCNTNNNAACNGNNNCNCNNANNNNNNNGNNNNNNNNNNNNNNNNNCCNNNGNNNNTT NNAGACNANTNTNNNNGNNNANNNNNNNNNNNNNNNNNNNTNNNNTGANTNGNNNNNNNNGNNNGNNNNNNNNNNNNTNNNGGNNNGNNNNNNNNNNNNNNANGNNNNNGACNTGCTNGNCATNNTCT NGGNNNNNNNTNANNNCNANAANNNTNNNNNNCANANNANTANCNTGNNNNNNNNNNNNTNGAGNGNNANNNNNNNNNNNNTCNCNNNANNNNNGGNNGCNCCTTTNACNANNNNNNNNNNNNTNANN NNNTNCTNNNNNANNNNNNTAAGNNNNNNCNTNNNNACANNNNNNNCGNANANCANNNNNNNNNNNNNNNNGGGNNNNNNGGGCNGNANNNNNANNNANNNNNNATGNTTNNNCNNNNNNNNNNNNNN GNNNNCTNTNNTGNNNNNNNNNNGGCGCTNANNNNAGNNNNNNNNGNCNNTNNNNNNNNNNNNNNNNNNNNNNNNNCNNNNNNNNNAAANTATNNNNNNNNNNNNTGGTNGNNNNAANNNNGTNNNAN NTNNNNTGGNGTNNNNNNCNCNNNTNTTNNGANGANCNNNNNNNNNNNNNNNGNNNNTNGNNNGGNCTTNNNNCNANNCNGNNNNNNNAANANTNNNNNGANNNNANNNANCNANNGNTTNCNNNNNN CNAANGGANGNNNNNANNNNGTNNTNNNNNAGNNNNNNNNCNNNGNNNGNNNANNANNANNNNNNNCNNNNT

Sample ATP2_mtptparev >ATP2-mtptparev 1283 56 93 0.05 NNNNNNNNNNNNNNNNGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGAGCGCANATTCGATAACCGCGAAAACTTCTTCAAAGTCAGAGTGGTCGCCGTAGTACGGGTCTTCAACGTCCAGAGCGTG GGTACCAGAACGCGGGTCGAAAGAACGCAGCATACGAACACGTGCCGCTTCAACACCCAGTTGGCGGAGGAGACGCGCATGGTTACGGTCGAGCGCAACCAGCAGGTCCGCTGCCAGGTGTTCGGTAC CAACCTGCGCCGCACGGNGGTCGGTCGGGTAGCCATGGGCACGCANAACACCGGCAGCGCGTCGTCCGCGCAAGAACCAACGTGCCAGTTGCCGGTACCTGCAGAGGTAACACGAACCGCGTCACCCA NACCACGGTGACGGAGCTGCTGCGCGAACATTTTTTCCGCCATCGGAGAACGGCAGATGTTACCGGTGCAAACGAAGGTAACGTGCAGCGGGTCANACATGGATTGGAAGTACAGGTTCTCGGNACCC AGATCTACACCAGAAGAATGATGATGATGATGGTGCATATGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAGGGGAATTGTTATCCGCTCACAATTCCCCTATAGTGAGTCGNATTAAT TTCGCGGGATCGAGATCTCGATCCTCTACGCCGGACNCNTCGTGGCCGGNNTCACCGGCGCCACAGGTGCGGTTGCTGGCGCCTATATCNNCNACATCACCGATGGGGAAGATCGGGCTCGCCACTTC NGGCTCATGAGCGCTTGTTTCNGCGTGGGTATGGNGGCAGNNCCCGTGGNCGGNNGACTGTTGGGCGCCNTCTCCTTGCATGCANCNTTCCTTGCGGCGGNGGNGCTCAACNGCCTCAACCTACTACT GGGCTGCTTCCTAATGCANGGAGTCGCATNANNGNAGAGNGNCNAGATCCCNGANNCNNTCGAATGGCGNAAANNCNTTTCGCGGNATGGCATGATANNNNCNNNAANANANNNANTNNGGNGNNTGA ANGNGAANNNAGTAACNTNNTNNNATGNNNNNNANTNNGNNNNNNNNNNNTNNTNNNANNNNNCCNNNNNGNNNNNNNNNNNNNNNNNTTNCNNNNAAACGNNNGNAAANNNNNNNCNNNNNNGNNNN NNNNNNNNNNNCCNNNNNNNGNCNNNNNNNGGNNNNNANNNNNNNNNNNTGANNNNNNNNNNCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNANNNNNNNNNCNNNNNNNNNNNNNNNNNNA NNN Sample ATP3_mtptpafor >ATP3-mtptpafor 869 869 0 0.05 NNNNNNNNNNNNNNNNNNNNNNANNNNNNNNNNNNNAACNNNNNNNNNANNNNNTCTCNNNNNNNNNNTNCNNNGNNNTNNNNGANNNNNNNNAACCCNNNNGNNNNGNCNANNNNNCCNNNTTGCNG AACTCTTCNNATTGGTGGANGTAAGCNGANTCNNANNGCTNACNTNNTGNTTNCNAAGNAGGCGCTCTANAANCTGANCTNTACACCCANNCANNGGGNNNNNNNGGACNNNNNNNNNGNACCTCNNC CTTANNNNGGNTGCNCNCTNNNNNNNTGANCTNNTAGCCCATATNGNNNNNNNACCTNGTNNNANNNNCTNNNNTGANTCNNNANNGACNNCNGNNGACCNNNGNNNTNNNCGNNANTCCTATNANGN NNNNNCGTCNGNNNGNCTNCTNGANAGGANGTGATCCANCNNCNNNTTNNNNNNCNGNTNNNNTGTNACGACTNCNCCCNANNNNNNNNCNNTGCNNNCNGCGCCCTCCTGNNTAANGTNAAAGNANT GACNNNNGNNNTGNNNANNNNNNNTNGNNNNNNNNTNANGTGNNNNNCAANGNNNNNNNACNTNTNNNNNNNNNGNNGCNNNCNTGCNANTNCNANCNATTCNNNNNTNNNNNNNGCNANNNNNNNNN NGNNNNCTNNNTNNNNCCNCNNNNNNNGNNNNANNTCNNNNNNCANCNGNTGGNTGCCNNNNNNNNCNCNNCNTTGTANNNACNNNTNNNNNNNNNGACNNNNNGGNNATGATNACTNGNNNTCNTCC CNACNNTNNNNNNGNTGNCCCCNGCANTCTCCTNNNNNGNTCNNNTNNNTGATGNNAACTAANGACAANNNNTGNGNTANNNGCGGNNTNNANCNCNNNTN

Sample ATP4_mtptparev >ATP4-mtptparev 1218 50 633 0.05 NNNNNNNNNNNNNNANGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGAGCGCAGATTCGATAACCGCGAAAACTTCTTCAAAGTCAGAGTGGTCGCCGTAGTACGGGTCTTCAACGTCCAGAGCG TGGGTACCAGAACGCGGGTCGAAAGAACGCAGCATACGAACACGTGCCGCTTCAACACCCAGTTGGCGGAGGAGACGCGCATGGTTACGGTCGAGCGCAACCAGCAGGTCCGCTGCCAGGTGTTCGGT ACCAACCTGCGCCGCACGGTGGTCGGTCGGGTAGCCATGGGCACGCAGAACACCGGCAGCGCGTTCGTCCGCGCAAGAACCAACGTGCCAGTTGCCGGTACCTGCAGAGGTAACACGAACCGCGTCAC CCAGACCACGGTGACGGAGCTGCTGCGCGAACATTTTTTCCGCCATCGGAGAACGGCAGATGTTACCGGTGAAACGAAGGTAACGTGCAGCGGGTCAGACATGGATTGGAAGTACAGGTTCTCGGTAC CCAGATCTACACCAGAAGAATGATGATGATGATGGTGCATATGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAGGGGAATTGTTATCCGCTCACAATTCCCCTATAGTGAGTCGTATTA ATTTCGCGGGATCGAGATCTCGATCCTCTACGCCGGACGCATCGTGGCCGGCATCACCGGCGCCACAGGTGCGGTTGCTGGCGCCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACT TCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCNNNCCCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCGGCGGNGGTGCTCAACGGNCTCAACCTACTAC TGGGCTGCTTNCTAATGCNGNNGTCGCATNNNNANAGCGTCNAGNTCCNNNACNNCATCGAATGGCGCAAAANCNTTCGCNGNATGGCATGATANCNNCNNNNNANAGTNANNNNNNNGNGAATGTGA AANNAGTANNTNNNCNATGTCNNNNANTNNNCNNNNTCNNTNTNNNCNNNCCNNNNNNNANNNNNNNCNNNNTNNTNNNNNANNNGGAAAANNNNNNGNNNNGNNNNNNTNANNNNNNNNNNNNNNNN NNNNACNNNNNNNAANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNANNNNNNNNNGNNN __**Analysis:**__ Sequencing results showed really high identity coverage percentage, but it was still consider unsuccessful cloning. The identity needs to be 100% in order to be considered a positive clone. There could have been silence mutation, mismatches and/or deletion. Both samples' forward DNA sequences showed a lot of N's, so there could have been error in the reverse primer used. In the future, I will try pLIC-for and pLIC-rev primers to prep my sample for DNA sequencing.

- Cohesive End Generation on PCR Inserts and Accepting Vector
Notes on protocol: 10X T4 DNA Polymerase Buffer is NEB Buffer 2

- Annealing and Transformation
Tube A: 2 ul of Accepting Vector + 4 ul of PCR Inserts Tube B: 1 ul of Accepting Vector + 9 ul of PCR Inserts Tube C: 2 ul of Accepting Vector + 7 ul of PCR Inserts

**- Transformation of competent cells for plasmid prep of pNIC-Bsa4**
Colony was put into 160 ml of LB and was shake high at 37C shaker at rpm of around 200-350 to grow up overnight for 16hrs.

**- Miniprep**
__**Analysis:**__ pNIC-Bsa4 was prep by cutting out sacB gene. PCR-cleanup was performed to remove excess primers and dNTPs from the PCR product. Cohesive end generation with T4 was done to insert dCTP onto PCR Inserts and dGTP onto accepting vector. Next, the plasmid was cloned by inserting it into E.coli DH5α to make more copies. Finally, the sample was prepared by miniprep with many cycles of centrifuging in order to send to DNA sequencing. The nanodrop spectrophotometry of sample 2A’ for cloned plasmid into E.coli DH5α is about 30.73 ng/μl and sample 2B’ is about 20.25 ng/μl. Cloning was successfully completed. The next step is to submit positive clones to DNA sequencing facility. If that is successful, I can move on to protein expression, purification, and characterization.

**11/14 - Cleaned PCR products of MtPtpA**
__**Analysis:**__ I learned my mistake from the first two times of PCR-clean up. I used the correct Elution Buffer for the very last step this time. The concentration yielded from nanodrop spectrophotometry showed high concentration from both samples. Sample D’ is about 266.8 ng/μL and sample F’ is about 273.8 ng/μL. Primary, secondary PCR were successfully done when I altered the amount of the ingredients I put into the reaction tube. Also, temperature gradient worked really well for secondary PCR. The banding pattern resulted at around 500bp, which is closed to 492bp of my gene of interest (MtPtpA). The concentration yielded was also really high. The next step is to do preparation of pNIC-Bsa4 as accepting vector and do PCR clean up for it. After that, cohesive end generation will be done for both the PCR inserts and accepting vector in order to be ready for annealing and transformation.

**11/14 - PCR Squared**
====== __**Analysis:**__ I used secondary PCR reactions of samples D’ and F’ to do PCR squared. I used the annealing temperature of 57.5°C for the thermocycler cycling conditions. The nanodrop spectrophotometry resulted in really high concentration for both samples. Sample D’ is about 675.2 ng/μL and sample F’ is about 456.15 ng/μL. Also, the gel electrophoresis check of both samples showed distinct band pattern around 500bp.

**11/08 - Secondary PCR**
__8 different samples of Secondary PCR were made. __ 10 μl of 5X rxn buffer and 5 μl of 2 mM were used for all the samples. 1 μl of template = primary PCR rxn (from 1 ° PCR A) 1 μl of 20 μM F primer 1 μl of 20 μM R primer 0.5 μl of Q5 hotstart Polymerase (1U/μl) 31.5 μl of dH2O
 * //Sample A, C, E //**, **//G//**

2 μl of template = primary PCR rxn (1 μl from 1 ° PCR A and 1 μl from 1 ° PCR C) 2 μl of 20 μM F primer 2 μl of 20 μM R primer 1 μl of Q5 hotstart Polymerase (1U/μl) 31.5 μl of dH2O
 * //<span style="font-family: Arial,sans-serif; font-size: 10pt;">Sample B, D, F, H //**

__**Analysis:**__ I used primary PCR reactions of samples A and C to do secondary PCR. This time, I also tried temperature gradient. My temperature ranges from 55°C to 66°C. I made 8 different samples (A’-H’) of secondary PCR. After checking my result with gel electrophoresis, all 8 the samples showed band patterns. The size of the DNA is around 500bp, which is close to the 492bp of my gene of interest (MtPtpA).

**11/07 - Primary PCR**
__Six different samples of Primary PCR were made.__ 10 μl of 5X rxn buffer and 5 μl of 2 mM were used for all the samples. //**Sample A:**// 2 μl of oligo mix (new) 1 μl of Q5 hotstart Polymerase (1U/μl) 32 μl of sterile dH2O //**Sample B:**// 1 μl of oligo mix (new) 1 μl of Q5 hotstart Polymerase (1U/μl) 33 μl of sterile dH2O //**Sample C:**// 2 μl of oligo mix (old) 1 μl of Q5 hotstart Polymerase (1U/μl) 32 μl of sterile dH2O //**Sample D:**// 1 μl of oligo mix (old) 1 μl of Q5 hotstart Polymerase (1U/μl) 33 μl of sterile dH2O //**Sample E:**// 1 μl of oligo mix (new) 0.5 μl of Q5 hotstart Polymerase (1U/μl) 33.5 μl of sterile dH2O //**Sample F:**// 1 μl of oligo mix (new) 0.5 μl of Q5 hotstart Polymerase (1U/μl) 33.5 μl of sterile dH2O

__<span style="font-family: Arial,sans-serif; font-size: 10pt;">Thermocycler cycling conditions used (20 cycles) __ <span style="font-family: Arial,sans-serif; font-size: 10pt;">1) 98 ° <span style="font-family: Arial,sans-serif; font-size: 10pt;">C for 30 sec <span style="font-family: Arial,sans-serif; font-size: 10pt;">2) 98 ° <span style="font-family: Arial,sans-serif; font-size: 10pt;">C for 8 sec <span style="font-family: Arial,sans-serif; font-size: 10pt;">3) 58 ° <span style="font-family: Arial,sans-serif; font-size: 10pt;">C for 20 sec (annealing temperature) <span style="font-family: Arial,sans-serif; font-size: 10pt;">4) 72 ° <span style="font-family: Arial,sans-serif; font-size: 10pt;">C for 25 sec/kb <span style="font-family: Arial,sans-serif; font-size: 10pt;">5) go to step 2 and repeat 19 times <span style="font-family: Arial,sans-serif; font-size: 10pt;">6) 72 ° <span style="font-family: Arial,sans-serif; font-size: 10pt;">C for 2 min <span style="font-family: Arial,sans-serif; font-size: 10pt;">7) 4 ° <span style="font-family: Arial,sans-serif; font-size: 10pt;">C for infinite

__**Analysis:**__ I did primary PCR again for the second time. I altered the amount of oligo mix and Q5 hotstart Polymerase that I put into the solution tube. I also did not dilute the Q5 hotstart Polymerase. I made six different samples (A-F) of primary PCR. From what I found, the ones that worked the best were the one with 2μl of oligo mix and 1μl of Q5 hotstart Polymerase. The smear patterns were more distinct and visible.

- Cohesive End Generation on PCR Inserts and Accepting Vector
Notes on protocol: 10X T4 DNA Polymerase Buffer is NEB Buffer 2

- Annealing and Transformation
Tube A: 2 ul of Accepting Vector + 4 ul of PCR Inserts Tube B: 3 ul of Accepting Vector + 6 ul of PCR Inserts Tube C: 2 ul of Accepting Vector + 6 ul of PCR Inserts Tube D: 3 ul of Accepting Vector + 4 ul of PCR Inserts

**- Transformation of competent cells for plasmid prep of pNIC-Bsa4**
Colony was put into 160 ml of LB and was shake high at 37C shaker at rpm of around 200-350 to grow up overnight for 16hrs.

__**Analysis:**__ Cloning was failed, there were no colonies on the plates after two days. During the annealing and transformation steps, after incubated on ice for 30 minutes, I forgot to heat shock for 30 seconds in 42C water bath before adding 100 ul of pre-warmed SOC to each tube. For next time I will remember to heat shock and I should not parafilm the plates while incubating upside down overnight at 37C for 1 day.

1162014- Fantastic Work!

**10/29 -** **PCR Cleanup for the gene insert (MtPtpA) Trial 2**
__**Analysis:**__ PCR clean-up was done to purify the solution. One thing that was done differently from the first trial of PCR cleanup was the use of elution solution last. The resulting concentration was about 59.15 ng/ul, which is higher than 35.85 ng/ul, the concentration of PCR cleanup trial 1. The next step is to do cohesive end generation for both PCR insert and accepting vector, then annealing and transformation will be done right after.

**10/28 - PCR** **Squared Trial 2**


__**Analysis:**__ PCR squared was done again from the already prep secondary PCR. The nanodrop spectrophotometry was about 479.05 ng/ul, which is really high. Gel electrophoresis shown on figure 1 has only smears of the four samples of PCR squared and light bands at around 500 base pairs. The distinct bands was not seen like from the first trial of PCR squared.

**10/24 - Preparation of pNIC-Bsa4 as Accepting Vector and PCR Cleanup for pNIC-Bsa4**
======

**PCR Cleanup for the gene insert (MtPtpA) Trial 1**


__**Analysis:**__ In preparation of pNIC-Bsa4, SacB gene was cut from the pNIC-Bsa4 because with the presence of sucrose, SacB will cause cell death. PCR cleanup was performed after removing the SacB gene. Three bands resulted from the gel electrophoresis check shown on figure 1, the two bands at around 5000 bp and 2000 bp are matched with the result from NEBcutter's virtual gel. PCR cleanup was also done for the PCR squared product of MtPtpA. The concentration resulted os 35.85 ng/ul was much lower than before doing PCR cleanup. The last solution used to elute the solution could be the potential problem. 10 mM Tris was diluted from the already made 100mM Tris, so the Tris solution could have been made incorrectly in the first place.

**10/16 - PCR Squared Trial 1**




__**Analysis:**__ PCR squared was done to amplify the template of the secondary PCR reaction to create a large yield of the gene MtPtpA. Four samples were made with the concentration around 387.5 ng/uL. The gel electrophoresis check for all four samples shows a clear distinct band at around 500 bp.

**1****0/13 -** **pNic28-Bsa4** **Plasmid Midi-Prep Trial 2**
__**Analysis:**__ The different washes and filters processes were done to prep the accepting vector of pNic28-Bsa4 for the insertion of the CDS of MtPtpA. The accepting vector DNA was isolated from the other unnecessary cell parts. The concentration resulted from nano-dropping was 46.3 ng/uL which is not so high. The isolated pNic28-Bsa4 is to be used next in the Restriction Enzyme Digest step.

982014- Outstanding work don't forget captions for your pictures.

**1****0/10 - Secondary PCR trial 2**
__**Analysis:**__ The gel check for secondary PCR on lane 2 showed no bands or smears. On the other hand, secondary* PCR of the template of secondary PCR reaction resulted in a successful gel check with dark band pattern at 500 bp. The next step is to do PCR squared to make more of the DNA made from secondary* PCR.

Note the different between secondary PCR and secondary* PCR:
 * secondary PCR used template from primary PCR reaction with the mixture consisted of oligo mix only.
 * secondary* PCR used template from secondary PCR reaction with the mixture consisted of oligo mix and forward & reverse primers.

**1****0/09 - Primary PCR Trial 2 and Secondary PCR trial 1**
__**Analysis:**__ Lane 3 was the gel check for primary PCR, but no smears or bands were shown. Lane 5 resulted in smears and bands patterns (1st band - 700 bp and 2nd band - 500 bp) from the gel check of secondary PCR. The secondary* PCR of the template of secondary PCR reaction is needed to amplify the full length of DNA, because the smears on lane 5 are presented but not distinct band.


 * Note of the secondary PCR: Instead of adding the template from the primary PCR reaction to the secondary PCR mixture, oligo mix was added.

**10/03 - Primary PCR Trial 1**
__**Analysis:**__ This first trial of primary PCR was resulted in neither smears or banding patterns. This could be resulted from inadequate amount of blue juice, 2 ul for 50 ul of the mixture in the tube. As a result, it was to light to show up on the gel. Redo of primary PCR is necessary in order to move into secondary PCR.

**10/02 -** **pNic28-Bsa4** **Plasmid Midi-Prep Trial 1**
__**Analysis:**__ The different washes and filters processes were done to prep the accepting vector of pNic28-Bsa4 for the insertion of the CDS of MtPtpA. The accepting vector DNA was isolated from the other unnecessary cell parts. During the process of pressing the plunger to push the solution through, the QIAprecipitator was leaking, so this could contributed to contamination of the DNA and some of the DNA might escaped out. The isolated pNic28-Bsa4 is to be used next in the Restriction Enzyme Digest step.

**09/29 - PCR Primer Design Tails for pNic28-Bsa4 Cloning**
Forward Primer: 5’ TAC TTC CAA TCC ATG TCT GAC CCG CTG CAC GTT AC 3’ Reverse Primer: 5’ TAT CCA CCT TTA CTG TTA AGA CGG ACC GTT ACG CG 3’ __**Analysis:**__ A virtual circular sequence was created from NEB cutter to represent pNIC28-Bsa4 plasmid with BsaI's cuts. A virtual gel was also generated to show the gene segments of BsaI on the plasmid. On the pNIC28-Bsa4 plasmid sequence, BsaI was removed and the CDS of MtPtpA was inserted instead for cloning purpose. There are 492 base pairs on the CDS including the start and stop codons. This virtual gel can be used in Restriction Enzyme Digest to verify with the gel elctrophoresis result.

9232014- Great work

**09/12 - PCR Primer Design for Primer Over Assembly PCR**
12 oligonucleotides need to be synthesized

1 ATGTCTGACCCGCTGCACGTTACCTTCGTTTGCACCGGTAACATCTG 47 2 CAGTTGTTGCGCGAACATTTTTTCCGCCATCGGAGAACGGCAGATGTTACCGGTGCAAAC 60 3 AATGTTCGCGCAACAACTGCGTCACCGTGGTCTGGGCGACGCGGTTCGTGTTACCTCTGC 60 4 GCTCATCCGCGCAAGAACCAACGTGCCAGTTACCAGTACCCGCAGAGGTAACACGAACCG 60 5 TTCTTGCGCGGATGAGCGCGCAGCCGGTGTTCTGCGTGCGCATGGTTACCCGACCGACCA 60 6 CAGGTCTGCCGCCAGGTGTTCGGTACCAACCTGGGCCGCACGGTGGTCGGTCGGGTAACC 60 7 CCTGGCGGCAGACCTGCTGGTTGCGCTCGACCGCAATCACGCACGTCTCCTGCGCCAACT 60 8 GTCGAAAGAACGCAGCATACGAACACGTGCCGCTTCAACACCCAGTTGGCGCAGGAGACG 60 9 GTATGCTGCGTTCTTTCGACCCGCGTTCTGGTACCCACGCGCTGGACGTTGAAGACCCGT 60 10 TAACCGCGAAAACTTCTTCAAAGTCAGAGTGGTCACCATAGTACGGGTCTTCAACGTCCA 60 11 TTTGAAGAAGTTTTCGCGGTTATCGAATCTGCGCTGCCGGGTCTGCACGACTGGGTTGAC 60 12 TTAAGACGGACCGTTACGCGCCAGACGTTCGTCAACCCAGTCGTGCAG 48

__**Analysis:**__ The amino acid sequence of MtPtpA was input into http://helixweb.nih.gov/dnaworks/ to create 12 oligonucleotides of forward and reverse primers for PCR synthesizing and amplifying the CDS, so that it can be inserted into a cloning (or expression) vector. These oligonucleotides were designed based on the amino acid sequence and have overlapping ends.

**09/08 - Day 2 - Transformation of competent cells for plasmid prep of pNICBsa4**



 * 09/09 - Day 3 - Transformation of competent cells for plasmid prep of pNICBsa4**


 * __Analysis:__**

=
The pellets of sample 1 and sample 2 were obtained after spinning down and each pellet consists of DH5alpha transformed bacteria with pNIC-Bsa4. These pellets are stored in the -20C fridge to be use in the purification process to clean out foreign elements.======


 * 09/08 - Day 1 - Restriction Enzyme**


 * 09/10 - Day 2 - Restriction Enzyme**


 * __Analysis:__**

=
The result from the gel electrophoresis resembled the result from the virtual gel from the Analyzing Sequence Lab. This technique can later be used in my own project to cut the gene to move into an expression plasmid.======

======
 * 09/16 - PCR Protocol**
 * __Analysis:__**

=
Only sample A (0.3 ng of 1:10000) and sample C (30 ng of 1:100 dilution) showed the results of DNA migration from the gel electrophoresis, so there could have been error with the 1:1000 dilution for sample B did not migrate. The next step is to do PCR Squared to amplify DNA from this PCR.======

**08/29 - Submitting DNA to DNA Sequencing Facility**



 * __Analysis:__**

=
The technique of nanodrop spectrophotomer was done to verify that the plasmid obtained was the correct plasmid to be used in the next step. The results after two trials of nanodropping show that the concentration is within the range of 100-500 ng/μl for pGBR22 plasmid, which is the correct plasmid. ======


 * __Analysis:__**

=
A sample of 12μl was prepared from 4μl of 2.2μM primer (reverse), 1.26μl of pGBR22, and 6.8μl of nanopure H2O. The result after the sample was sequencing shows a sequence of DNA that will be later used in computer lab for further analyzing of the DNA sequence of pGBR22 plasmid.======


 * 09/02 - Day 1 - Transformation of competent cells for plasmid prep of pNIC-Bsa4**


 * __Analysis:__**

=
After the transformation of DH5alpha cell with 50ng of pNIC-Bsa4 plasmid with Kanamycin was plated on LB agar plates overnight in 37 ° C incubator, the result shows that the plate with 10μl of the sample has less colonies of bacteria. On the other hand, the plate with 50μl of the sample has more abundant colonies. The size of each colony in the 10μl is bigger than of the 50μl. The sizes difference could be resulted from the competition for nutrients and space on the LB agar plates.======