Janice+K.

Fall 2012 __**Inhibition Assay FtHAP (Second Attempt) Compound 5154121**__
 * Week 13**

For the second run, the concentration of the diluted enzyme was kept the same, but only 22.5 ul of the enzyme in Tris-acetate instead of 30 ul. The absorbance lowered so the spectrophotometer can give a better reading. As seen in the Excel graph, the absorbance measured displayed decreasing absorbance as the concentration of the inhibitor increased. This inhibition assay demonstrates a possibility of the compound effectively inhibiting the FtHAP_DXR. In order to confirm the findings, more inhibition assays should be completed. Figure 2: Inhibition Assay- Absorbance at 420 nm vs Inhibitor Concentration (um) for FtHAP second run Figure 1: Inhibition Assay- Absorbance at 420 nm vs Inhibitor Concentration (uM) for FtHAP first run -Completed first run for CB_kin library __**Enzyme Assay FtHAP (First Attempt) Compound 5154121**__

The graph was supposed to show steady increase in absorbance as the concentration of the enzyme were raised. Although the last two measurements at high enzyme concentration displayed significantly higher absorbance, the data points did not show any pattern. The sources of error may have wrong dilutions of NaOH and stock enzyme. Moreover, wrong measurements of the reagents added may have caused the sporadic pattern. This experiment will need to be repeated before continuing on the inhibition assay.

Figure 3: Absorbance at 420 nm vs Enzyme Concentration (uM) for FtHAP Figure 1: Absorbance measured at 420 nm for FtHAP with various enzyme concentrations

-DNA sequencing result for FrTu_DXR -reconcentrated to 1.00 mL of FtHAP after FPLC -added glycerol & stored at -20C
 * came back with just pNIC-Bsa4 vector sequence or tons of N's
 * __Nanodrop result for FtHAP after reconcentration__**

The nanodrop results for FtHAP after reconcentration were measured at 2.26 and 2.25 mg/mL. __**Nanodrop result for FtHAP before reconcentration**__

The nanodrop result for FtHAP before reconcentration was measured at 0.47 mg/ml after FPLC.
 * __Fast Protein Liquid Chromatography (FPLC) Result for FtHAP__**

The FPLC showed that tubes 32-38 contained the FtHAP protein.. The buffer used for the process contained 100 mM Tris, 150 mM NaCl, pH 8.0. The absorbance was measured at 280 nm. After initial concentration, there were 1.00 mL of FtHAP used for FPLC. The initial data collection demonstrated a carry over from the previous FPLC with another protein, but it stabilizes soon as the buffer runs through the machine. The large bump may or may not located at the right size, but it was determined to be close enough to continue. The second bump out of the three represents the location of the target protein.These selected tubes were saved and reconcentrated to 1.00 mL. Glycerol was added to the sample and stored at -20C.

112612 - Good. Dr B
 * Week 12**

-concentrated Elution #1 & FPLC 11/20/2012 -Protein Characterization for FtHAP (will dry gel later) -prepared samples for DNA sequencing to check for positive clones 11/19/2012 Protein Purification Elution #1 Nanodrop Reading measured at 1.78 mg/ml (FtHAP in BL21 (DE3)) Protein Purification Elution #1 Nanodrop Reading measured at 0.25 mg/ml (FtHAP in BL21 (DE3))
 * __Nanodrop Result after Protein Purification FtHAP with Sadhana's Protocol__**

Nanodrop result after miniprep measured at 103.0 ng/nL (FrTu_DXR Sample 1) Nanodrop result after miniprep measured at 124.1 ng/uL (FrTu_DXR Sample 2) Nanodrop result after miniprep measured at 91.3 ng/uL (FrTu_DXR Sample 3) Nanodrop result after miniprep measured at 90.8 ng/uL (FrTu_DXR Sample 4) Nanodrop result after miniprep measured at 319.7 ng/uL (FrTu_DXR Sample 5) Nanodrop result after miniprep measured at 77.1 ng/uL (FrTu_DXR Sample 6) Nanodrop result after miniprep measured at 92.8 ng/uL (FrTu_DXR Sample 7) Nanodrop result after miniprep measured at ng/uL (FrTu_DXR Sample 8) Good - Dr. B 11/19/12 -growing FtHAP with Sadhana's protocol -Spun down 8 samples after 16 hours of growth & moved master plate from 4C to -20C (11/13/2012) at 11 am -Picked 8 colonies to grow for 16 hours & made master plate (11/12/12) 7 pm -Attempted cloning for FrTu_DXR (11/10/12) will check plates on Monday Francisella Tularensis_DXR transformation plate #1 (3ul of Accepting + 10 ul of PCR insert) Francisella Tularensis_DXR transformation plate #2 (5ul of Accepting + 5 ul of PCR insert) Francisella Tularensis_DXR transformation plate #3 (5ul of Accepting + 7 ul of PCR insert)
 * __Nanodrop Result after Miniprep for FrTu_DXR (First Attempt)__**
 * Week 11**

I picked two colonies from each plate to make the master plate. There were little bit more colonies present on the plates than targeted 20 colonies. After letting it grow for 16 hours, the samples were spun down. The master plate was moved to 4C while the 8 samples are now stored in -20C. The midiprep kit will be used to extract and purify the DNA. I am hoping to the send the samples to DNA sequencing by Friday.

Although my transformation plates did not previously grow during my first two attempts, I accidentally left my pNIC-Bsa4 + PCR insert + DH5alpha sample in the 37C shaking incubator for 2.5 hours instead of 1 hour. I was worried that it might have overgrown, but I was able to see growth on my transformation plate. Although I am a little skeptical about my plates, I will continue on to identifying the positive clone.

__**Secondary PCR WBM_DXR (First Attempt)**__ Lane 2: 100 bp ladder Lane 3-10: Secondary PCR for WBM_DXR

Although my primary PCR for WBM worked, I'm not sure why I have smear bands rather than bright bands for my secondary PCR samples. I might just continue to PCR squared to see what is going on. It also might have been the gel since my 100 bp ladder looks smeared. I might try adjusting the annealing temperature to see how the change affects my samples.

-Homology model prepared for virtual screening using Hermes -First virtual screening run using homology model for FrTu with cb_306 -Made oligo mix for WBM_DXR

Lane 2: 100 bp ladder Lane 3,5,7,9- Primary PCR
 * __Primary PCR WBM_DXR (First Attempt)__**

I used 58C for the annealing temperature for the primary PCR for WBM. I think this temperature works perfectly since I got bright smears on the gel. I will continue on with Primary PCR #1 to make my secondary PCR samples.

Lane 2: 1kb ladder Lane 4: cut pNIC-Bsa4 (NEB) Lane 6: cut pNIC-Bsa4 (Fermentas)
 * Week 10**
 * __Cut pNIC-Bsa4__**

I just threw these samples away due to the extra bands. I think there is something wrong with my stock pNIC-Bsa4. I am making more using the staff pNIC-Bsa4. __**Homology Model**__

Modeled Residue Range: 2 to 385 Template: 3IIEA Sequence Identity: 45.844 QMEAN Z-Score: -1.994 QMEANscore4: 0.645

__**Primer Sequence**__ FOR: 5'- TAC TTC CAA TCC ATG TTC AAA AAA ACC-3' REV: 5'- TAT CCA CCT TTA CTG TTA GCC CAG AA-3' __**Primers for Plate**__ Lane 2: 1kb ladder Lane 4: cut pNIC-Bsa4 Lane 6: cut pNIC-Bsa4 Figure 1: Nanodrop result of PCR^2 eluted in 50ul of elution solution measured at 86.4 ng/ul Figure 2: Nanodrop result of PCR^2 eluted in 50ul of elution solution measured at 83.7 ng/ul Figure 3: PCR^2 Gel Check Lane 2: 100bp ladder Lane 3-10: PCR^2 samples
 * __Cut pNIC-Bsa4 used ECO311__**
 * __PCR^2__**

I started my cohesive end generation for PCR insert and accepting vector. After I placed the samples in the heat block, the temperature was little bit lower than the target temperature. I turned the knob slightly to adjust the temperature, but I ended up overshooting. When I came back to retrieve my samples, they were all evaporated (even though I placed another block on top of the lids). :( I ran out of my PCR^2 so I am making more for cloning.

Lane 2: 100bp ladder Lane 4: cut pNIC-Bsa4 from last weekend Lane 6: cut pNIC-Bsa4 from last weekend
 * __Cut pNIC-Bsa4 used ECO311 (made from last weekend) Gel Check (Threw it away)__**

I gel checked the cut pNIC-Bsa4 that I made from last weekend. Although the concentration was measured around 67.6ng/ul, there was a smear on top of the two lines. I ended up just throwing it away and made new cut pNIC-Bsa4 for tomorrow (Sunday). I also used the wrong ladder for the gel so I will make sure to use 1kb ladder next time.

102112 - Janice, be sure to use the newer T4 DNA Polymeraase and the newer tubes of dGTP and dCTP. Also, if you want to team up with Aldo and split the work some - you may be able to move faster. - Dr. B

I tried cloning twice this week, but nothing ended up growing on the transformation plates. I used the 37C shaking incubator in Welch for the cohesive end generation of PCR inserts and accepting vector. I also used the same Kan+Suc plate made by someone else from 8/28/2012 so I am considering on making my own or using plates that were made more recently.
 * Week 9 (10/15/2012-10/20/2012)**

__**Making more cut pNIC-Bsa4 and PCR fragment for next week**__ -will gel check my cut pNIC-Bsa4 on Monday (ran out of time on Saturday) Figure 1: Concentration of PCR^2 after clean up measured at 67.6 ng/ul Figure 2: Concentration of PCR^2 after clean up measured at 66.0 ng/ul
 * __Nanodrop results for PCR^2 samples__**

Mixing the binding solution along with the samples in a separate tube before transferring it to the PCR clean tubes for the spin down slightly raised my concentrations since I used the same secondary PCR sample to make the PCR^2 last week. __**Gel Check for PCR^2 samples**__ Lane 2: 100 bp ladder? Lane 3-10: PCR^2 samples

The 100 bp ladder did not show up on the gel. I remember taking out & returning the 100bp ladder back to the -20C, but I think I may have forgotten to actually use it. :) I was pretty sure it was the right size since I used the same secondary PCR sample for my previously PCR^2. There was no apparent contamination from the gel.

101612 - Janice, looks good. Good luck with the cloning steps! -- Dr. B Next week: -continue to Cohesive End Generation on PCR inserts and Accepting Vector -start on homology model (?) Lane 2: 1kb ladder Lane 4: cut pNIC-Bsa4
 * Week 8/9 (10/13/2012-)**
 * __Cut pNIC-Bsa4 (First Attempt) WORKED!!!!!__**


 * __Nanodrop Result for Cut pNIC-Bsa4__**

I changed the final volume to 50 ul. I also doubled all the required reagents. In order to have 2250ng of pNIC-Bsa4 plasmid, I added 41.74 ul of pNIC-Bsa4 sample. The sample was placed in 37**°**Cwater bath for three hours. During the PCR clean up, I accidentally eluted the cut pNIC-Bsa4 in 50 ul of elution solution instead of 30ul. This might be the reason why the concentration came out on a little low side.

100912 - Janice - GREAT JOB - We have new T4 DNA Poly that may work better for your cloning (new dGTP and new dCTP also) - Dr. B
 * Week 8 (10/8/2012-10/13/2012)**

__**Nandrop Result for PCR^2 WORKED!!!**__ Figure 1: Concentration of PCR^2 measured at 65.4 ng/uL Figure 2: Concentration of PCR^2 measured at 65.9 ng/uL The Lane 2: 100bp ladder Lane 3-10: PCR^2 from Secondary PCR #1
 * __PCR^2 (Third Attempt) using only Secondary PCR #1__**

The concentration of PCR^2 previously came out really low since I eluted with the wrong amount. I started from the primary PCR (#2) to make secondary PCR (#1). Then, I used the Secondary PCR (#1) to make PCR^2 samples. When I eluted in 50ul of elution solution for PCR clean up, the concentration of PCR^2 came out within range (65.9ng/uL).

dropped my gel :( will gel check it again on Monday __**Secondary PCR (Second Attempt)**__ Lane 2: 100bp ladder Lane 3: Secondary PCR #1(red) Lane 4: Secondary PCR #2 (red) Lane 5: Secondary PCR #3 (red) Lane 6: Secondary PCR #4 (red) Lane 7: Secondary PCR #1 (blue) Lane 8: Secondary PCR #2 (blue) Lane 9: Secondary PCR #3(blue) Lane 10: Secondary PCR #4(blue)
 * Week 7**
 * __PCR^2 (Third Attempt) using only Secondary PCR #1__**
 * made from Primary PCR #2 (orange)

The pNIC-Bsa4 vector (53.9 ng/ul) was blasted with the pNIC-Bsa4 FASTA sequence from Google Docs. I first took off some unnecessary N's from the beginning and end before comparing the sequences. Both the forward and reverse shows similarity to the original. However, there were some deletions noticeable. I am not sure whether the vector is still useable. Query coverage: 92% Max ident: 99% Query coverage: 91% Max ident: 99% -pNIC-Bsa4 vector sent to DNA sequencing- 10/02/2012 Week 6 __PCR^2 Nanodrop Results (2nd Attempt)__ Week 5** Made pNIC-Bsa4 vector & new PCR^2 I think the concentration of the pNIC-Bsa4 vector is high enough for cloning.
 * __pNIC-Bsa4 DNA Sequencing Analysis__**
 * __Forward pNIC-Bsa4__**
 * __Reverse pNIC-Bsa4__**
 * 100112 - Janice hmmm - not sure what is going on with PCR cleanup. Can't remember is you said you thought you knew. Hopefully better luck next time. - Dr. B**
 * Will continue to PCR cleanup
 * Need to sequence pNIC-Bsa4
 * __Making pNIC-Bsa4 vector for cloning__**
 * __PCR^2 (Second Attempt)__**

Lane 2: 100bp ladder Lane 3: PCR^2 for Secondary PCR #1 Lane 4: PCR^2 for Secondary PCR #1 Lane 5: PCR^2 for Secondary PCR #1 Lane 6: PCR^2 for Secondary PCR #1 Lane 7: PCR^2 for Secondary PCR #2 Lane 8: PCR^2 for Secondary PCR #2 Lane 9: PCR^2 for Secondary PCR #2 Lane 10: PCR^2 for Secondary PCR#2 The concentration of both samples were really low (8.8ng/ul and 7.2ng/ul). I already remade the PCR^2 samples from Secondary PCR #1 and #2. For next week, I will clean the samples up and measure the concentration again.
 * __Nanodrop__ __Results for both PCR^2__**

__**PCR^2 (First Attempt):**__ Proceeded to PCR Cleanup for Both PCR^2 samples Finished working on Virtual Screening due on Friday Will be moving onto PCR^2 using Secondary PCR #1 and Secondary PCR #2 Lane 2: 100bp Lane 3: Secondary PCR #1 (Primary PCR #2) Lane 4: Secondary PCR #2 (Primary PCR #4) Lane 5: Secondary PCR #3 (Primary PCR #2) Lane 6: Secondary PCR #4 (Primary PCR #4) Lane 8-10: Max's samples
 * Week 4- Janice, nice job! Be sure to make enough that you will have lots for cloning -- DR. B**
 * PCR^2 made on Thursday
 * Will gel check the samples next week
 * __Secondary PCR (First Attempt) WORKED!!! 9/20/2012__**

Bands are located around ~1100 & matches my gene size Secondary PCR #1 &2 were placed in a different thermocycler than Secondary PCR #3 & #4. The new thermocycler that I am using seems to bring out brighter bands even though I am using the same time and temperature from the PCR protocol. Lane 2: 100bp ladder Lane 3: Primary PCR #1 (red tubes)-Janice Lane 5: Primary PCR #2 (orange tubes) Lane 6: Primary PCR #3(yellow tubes) Lane 7: Primary PCR #4 (pink tubes) Lane 9: PCR^2-Aldo
 * __Primary PCR (Fourth Attempt) WORKED!!! 9/18/2012__**

Janice - is this with KOD or Q5? KOD Why do you think it is failing? I started using a different thermocycler. Now, it works better. What will you try next to make it work? I think making multiple samples of primary PCR helped since it increased my chances of having at least one working. - Dr. B 091812
 * Week 3-**

__Primary and Secondary PCR (Third Attempt)__ Lane 2: 1kb ladder Lane 3: Primary PCR Lane 4: Primary PCR Lane 6: Secondary PCR Lane 8: Secondary PCR

__Primary PCR with Q5 (Second Attempt)__ Lane 2- 1kb ladder Lane 3- Primary PCR- Aldo Lane 5 & 6- Primary PCR- Janice


 * Week 2**- Making new oligo mix

__Primary & Secondary PCR (First Attempt)__ Lane 2- 100bp ladder Lane 4- Primary PCR Lane 6- Secondary PCR
 * Week 1**- Finished PyMol refresher (posted on Google Docs)

Summer 2012 Lane 1: Skipped Lane 2: 1kb ladder Lane 3: pNIC-Bsa4 accepting vector- Janice Lane 4:Skipped Lane 5: pNIC-Bsa4 accepting vector- Aldo pNIC-Bsa4 sample after clean up
 * pNIC-Bsa4 cloning (Second Attempt)**


 * Choosing Restriction Enzyme for FtDXR_pNIC-Bsa4**

Elution #1 Elution #1 Elution #2 Elution #2 Sample 1: pNIC-Bsa4 cloning Mini Prep Sample 2: pNIC-Bsa4 cloning Mini Prep Sample 3: pNIC-Bsa4 cloning Mini Prep Sample 4: pNIC-Bsa4 cloning Mini Prep Sample 5: pNIC-Bsa4 cloning Mini Prep Sample 6: pNIC-Bsa4 cloning Mini Prep Sample 7: pNIC-Bsa4 cloning Mini Prep Sample 8: pNIC-Bsa4 cloning Mini Prep Lane 1: Skipped Lane 2: 100bp ladder Lane 3: Wrong Sample Lane 4: pNIC-Bsa4- Priya Lane 5: PCR^2 Lane 6: PCR^2 Lane 7: PCR^2 Lane 8: PCR^2 Lane 9: pNIC-Bsa4-Janice pNIC-Bsa4 sample after clean up __Second Attempt__ Midi Prep result for pNIC-Bsa4 grown in LB+Kan reading at 33.6 ng/uL and 32.6ng/uL Average:33.1 ng/ul
 * Protein Purification Nanodrop Results**
 * pNIC-Bsa4 Cloning Miniprep Results**
 * pNIC-Bsa4**
 * Midi Prep**

__First Attempt__ Midi Prep result for pNIC-Bsa4 grown in LB+Kan reading at 4.8 ng/uL Midi Prep result for pNIC-Bsa4 grown in LB+Kan reading at 4.2 ng/ul __Third Attempt__ Lane 1: Skipped Lane 2: 100bp ladder Lane 3: Sample 1 Lane 4: Sample 2 Lane 5: Sample 3 Lane 6: Sample 4
 * PCR^2**

__Second Attempt__ Lane 1: Skipped Lane 2: 100bp ladder Lane 3: Sample 1-Janice Lane 4: Sample 2 Lane 5: Sample 3 Lane 6: Sample 4 Lane 7: Skipped Lane 8: 100bp ladder Lane 9: Sample 1- Urvashi Lane 10: Sample 2 __First Attempt__ Lane 1: Skipped Lane 2: 100bp ladder Lane 3: Sample 1 Lane 4: Sample 2 Lane 5: Sample 3 Lane 6: Sample 4 __Fourth Attempt__ Lane 1: Skipped Lane 2: 100bp ladder Lane 3: Secondary PCR for Francisella Tularensis __Third Attempt__ Lane 1: Skipped Lane 2: 100bp ladder Lane 3: Secondary PCR for Francisella Tularensis Lane 4: Primary PCR
 * Primary and Secondary PCR**

__Second Attempt__ Lane 1: Skipped Lane 2: 100bp ladder Lane 3: Primary PCR for Francisella Tularensis-Janice Lane 4: Secondary PCR Lane 5: Skipped Lane 6: Skipped Lane 7: Primary PCR- Max Lane 8: Secondary PCR __First Attempt__ Lane 1: Skipped Lane 2: 100bp ladder Lane 3: Primary PCR- Alex Lane 4: Secondary PCR Lane 5: Primary PCR- Max Lane 6: Secondary PCR Lane 7: Primary PCR-Janice Lane 8: Secondary PCR __Fourth Attempt for pmCherry__ Lane 1-Skipped Lane 2: 100bp ladder Lane 3: R5 (1:100000) with GFP Lane 4: R4 (1:10000) with GFP Lane 5: R3 (1:1000) with GFP Lane 6: No DNA Lane 7: R5 (1:100000) with M13 Lane 8: R4 (1:10000) with M13 Lane 9: R3(1:1000) with M13 Lane 10: No DNA
 * PCR results for pGFP**

__Third Attempt__ Lane 1-Skipped Lane 2: 100bp ladder Lane 3: G5 (1:100000) with GFP Lane 4: G4 (1:10000) with GFP Lane 5: G3 (1:1000) with GFP Lane 6: No DNA Lane 7: G5 (1:100000) with M13 Lane 8: G4 (1:10000) with M13 Lane 9: G3(1:1000) with M13 Lane 10: No DNA __First Attempt__ Lane 1-Skipped Lane 2: 100bp ladder Lane 3: G5 (1:100000) with GFP Lane 4: G4 (1:10000) with GFP Lane 5: G3 (1:1000) with GFP Lane 6: No DNA Lane 7: G5 (1:100000) with M13 Lane 8: G4 (1:10000) with M13 Lane 9: G3(1:1000) with M13 Lane 10: No DNA

__ First Attempt __ *My samples did not run on the gel likely since there were some delay in putting them in the PCR after adding Taq __Second Attempt__
 * PCR results **
 * Transformation Efficiency**

Plate A: 3.47ul of pGBR22 on LB-Amp plate (counted 1600 colonies) Plate B: 1.74ul of pGBR22 on LB-Amp plate (counted 2000 colonies) Plate C: 4.34ul of pGBR22 on LB-Amp plate (counted 4000 colonies) Trial #1 260/280: 1.88 260/230: 2.30 Trial #2 260/280: 1.96 260/230: 2.53
 * Restriction Enzyme Digest **
 * Quantifying DNA using Nanodrop (pGBR22-purple protein) **

Average of Trial 1 & 2 260/280: 1.92 260.230: 2.415

http://helixweb.nih.gov/tmp/dnaworks_1338926014p1697_2055/LOGFILE.txt
 * Primer Sequence for 1-deoxy-D-xylulose 5-phosphate reductoisomerase (Francisella Tularensis subsp. Tularensis) **

code AANNNNNNNNNNNNGTGNNNANNNANNCACGCNNTCCNTCNNCGCGNCNNNGGNNCTCTCNNNTGNTNNNTTTGNNNNNN NNNGGNNNNNGCTNNGNNGATTGAAAGAGAAATATCTCANGTGTGANTCNNGTGCNTGCTCTGTTGGGGNGCTGTCNNNN GNNGGGTCNCTAAAGGANNATCCTGCGGANTTCNNNGGNGNTTNNTNNNTTTANNNNNNGNANNNNANNANGACNATNAN NNCCNNCCTCNANGGNNNNCNGCNNCTTANNTGNNATCNATNNTTATNNCNAACCCAANATNCATTCCTTCCNNCAACNN TTGNTNNGTANNCNNNGNTNNTNATNTNCANNNNNNGTTCTTGNNNGANANANGTANNNNNGAAATTNTNACTTANTNNA AANACNGGNNNNNNTNNNNNCNATGANNNNANNNNTCCNGNNNNNNNNNTCNNCTATGANNTAATNANNNGNGNNCNGNN ANATNNNCTNNNATGGANNNCNNNNNNNANGAAGNNNNNNNNNNGGGANNNNNNNNTNNNNNNGNNNNNNNCNATTNNNT TNNNGATANNNNANTCTTTANNAACNTTGAAGGAGGANNNATGATGTNACNNNNTNNNNNANTCTTACNNGCTNNNAAGC NNGNTAGGTNNNCACNNNNNNNNTACTGANNNNNNNNNNTATAANGNNTNNNANNNNNACNCANNNNNTAGANNANTGTG NATTAACNNANNTNTCAANNNNNNCCNCNNTTGCNCNCNCNATNNNCNTCANNNNNCNNCCCTNNCGANCGNNNGNGGNC ACNNCGCANNCNNTGNCGNGNATAATGNNNACNNTNNANTNNNNTNNNANNTTNGNNNGNNNNNNNNCTGNNNANNNNNC CNNNGGTNNANNANNTNNTNNAANNNNNNNNTNCTCCNCCNTNTNCNNNNNNNNCCTTCCCCCNNNNGGNNCNNNNNNNN NNNNNTCCNNNNGNNTANNNNNNNTANANNNNNGGGACGCTTNNNNNNCNNNNNNTNNCCCNGGAAGNNGNACNGGGNAA GGNNNNTNNNGNAGNNGGNNNNNTATNNNNNNNGNNTNNNNTNNNNTNN code code NNNNNNNNNNNGANNATAGAATACTCAAGCTATGCATCCAACGCGTTGGGAGCTCTCCCATATGGTCGACCTGCAGGCGG CCGCACTAGTGATTTTGATTGATTGAAGGAGAAATATCATGAGTGTGATCGCTAAACAAATGACCTACAAGGTTTATATG TCAGGCACGGTCAATGGACACTACTTTGAGGTCGAAGGCGATGGAAAAGGAAAGCCTTACGAGGGGGAGCAGACGGTAAA GCTCACTGTCACCAAGGGTGGACCTCTGCCATTTGCTTGGGATATTTTATCACCACTGTCTCAATACGGAAGCATACCAT TCACCAAGTACCCTGAAGACATCCCTGATTATGTAAAGCAGTCATTCCCTGAGGGATATACATGGGAGAGGATCATGAAC TTTGAAGATGGTGCAGTGTGTACTGTCAGCAATGATTCCAGCATCCAAGGCAACTGTTTCATCTACAATGTCAAAATCTC TGGTGTGAACTTTCCTCCCAATGGACCTGTTATGCAGAAGAAGACACAGGGCTGGGAACCCAACACTGAGCGTCTCTTTG CACGAGATGGAATGCTGATAGGAAACAACTTTATGGCTCTGAAGTTGGAAGGAGGTGGTTACTATTTGTGTGAATTCAAA TCTACTTACAAGGCAAAGAAGCCTGTGAGGATGCCAGGGTATCACTATGTTGACCGCAAACTGGATGTAACCAGTCACAA CAAGGATTACACATTTGTTGAGCAGTGTGAAATATCCATTGCACGCCACTCTTTGCTCGGTCATCACCATCACCATCACT AAAATCCCGCGGCCATGGCGGCCGGGAGCATGCGACGTCNGGCCCAATTCGCCCTATAGTGAGTCGTATTACAATTCACT GGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGNGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCG CCAGCTGGCGTAATAGCGANANGCCCGCANCGATCGCCCTTCCCANANTNCNCANCTNNNGNNATGNNNCNCCNGNANNG NNNATNNCNNGGCGGGGNNNGGNNNNNCNNNCANCGNGNCNNTANNNNNCNGNNNCNANCNNNNNNNTNNNTNNNNNNNN NNNNGCNNNGTNNCNNNTNNNNNNNNNNNNNNNNGGGNNNCNNNNNGNNNNANTNNNNTNNNGNNNNNGANNNNNNNNNN NNNNNNNNNNNGGNNTNNNNNNNNAACNNTTNCNN code
 * DNA Sequencing Result**
 * 061212 - So, Janice - this is DNA sequencing result of what plasmid? --Dr. B**
 * Forward:**
 * Reverse:**