Ramiro+R.


 * Could use more data and analysis -UM**

__**Week 13-15**__

What I will do:

Enzyme/Inhibition Assays Protein Expression, Purification, Characterization Make pNIC-BSA4 Cloning Finish Virtual Work Final Lab Report

Nanodrop of pNIC-BSA4 on 11/21/13. The yield was 19.3 ng/uL and at 260/280 (purity in relation to the protein) it was 1.96 and at 260/230 (purity in relation to other contaminates) it was 1.81.
 * Figure 25.**

__Analysis:__

The yield was low at 19.3 ng/uL. I will need to redo this and hope for better results. There are several possible errors that could have contributed to a low yield. I may have done the dilution of pNIC-BSA4 incorrectly. I also may have waited to long before putting the vector in the water bath. This is because I was waiting for it to heat up. Next time I will pre-heat the water bath.

Nanodrop of pNIC-BSA4 on 11/21/13. The yield was 19.9 ng/uL and at 260/280 (purity in relation to the protein) it was 1.96 and at 260/230 (purity in relation to other contaminates) it was 1.94.
 * Figure 24.**

__Analysis:__

The yield was low at 19.9 ng/uL. I will need to redo this and hope for better results. There are several possible errors that could have contributed to a low yield. I may have done the dilution of pNIC-BSA4 incorrectly. I also may have waited to long before putting the vector in the water bath. This is because I was waiting for it to heat up. Next time I will pre-heat the water bath.

Nanodrop of PCR Clean-up results on 11/20/13. The yield was 50.2 ng/uL and at 260/280 (purity in relation to the protein) it was 1.89 and at 260/230 (purity in relation to other contaminates) it was 2.04.
 * Figure 23.**

__Analysis:__

The yield was decent at 50.2 ng/uL. The 260/280 (1.89) and 260/230 (2.04) were also near their respective values. I will move on from PCR Clean-up and make pNIC-BSA4 tomorrow. I will begin my 1st cloning attempt.

__**Week 11-12**__

What I did:

PCR Clean up Make pNIC-BSA4 Continue Virtual Work

Nanodrop of PCR Clean-up results on 11/11/13. The yield was 24.3 ng/uL and at 260/280 (purity in relation to the protein) it was 1.82 and at 260/230 (purity in relation to other contaminates) it was -3.05.
 * Figure 22.**

__Analysis:__

The yield was very low and I will need to make another PCR Squared and repeat the results. The 260/280 is good since it was near 1.8, but the 260/230 was not near 2.1. This means there are other contaminates in the solution.

Nanodrop of PCR Clean-up results on 11/11/13. The yield was 17.3 ng/uL and at 260/280 (purity in relation to the protein) it was 1.88 and at 260/230 (purity in relation to other contaminates) it was -1.94.
 * Figure 21.**

__Analysis:__

The yield was very low and I will need to make another PCR Squared and repeat the results. The 260/280 is good since it was near 1.8, but the 260/230 was not near 2.1. This means there are other contaminates in the solution.

__**Week 9-10**__ 11/7/13 - Ramiro - try adding a small amount of DMSO to help prevent primer-dimer formation. - Dr. B

What I did:

Continue attempting Primary and Secondary PCR at different times and temperatures Do PCR Squared and Clean-up PCR Continue Virtual work Make pNIC-BSA4

Figure 20. PCR squared of Lane 4 Secondary PCR from figure 12. Lane 1 and 6 have the 100 base pair ladder and lanes 2-5 have the 4 PCR squared samples at different annealing temperatures. 11/4/13

__PCR Squared Temperatures__

98 degrees Celsius_ 2 minutes 98 degrees Celsius_ 20 Seconds ? degrees Celsius_ 20 Seconds ANNEALING TEMPERATURE (See Below) 72 degrees Celsius_ 20 Seconds Repeat 2-4, 25 cycles 72 degrees Celsius_ 2 minutes 4 degrees Celsius_ Infinite

__Annealing Temperatures for PCR Squared:__

Lane 2 (A) 65 degrees Celsius Lane 3 (B) 64.3 degrees Celsius Lane 4 (C) 56 degrees Celsius Lane 5 (D) 55 degrees Celsius

__Analysis:__

I used the sample from lane 4 in figure 12 because it had the least primer dimer bands. In this PCR squared the sample in lane 3 came with minimal brightness that was not my gene. I will move forward with this PCR squared to PCR clean up and see what yield I get.

Figure 19. PCR squared of Lane 3 Secondary PCR from figure 12. Lane 1 and 6 have the 100 base pair ladder and lanes 2-5 have the 4 PCR squared samples at different annealing temperatures. 11/1/13

__PCR Squared Temperatures__

98 degrees Celsius_ 2 minutes 98 degrees Celsius_ 20 Seconds ? degrees Celsius_ 20 Seconds ANNEALING TEMPERATURE (See Below) 72 degrees Celsius_ 20 Seconds Repeat 2-4, 25 cycles 72 degrees Celsius_ 2 minutes 4 degrees Celsius_ Infinite

__Annealing Temperatures for PCR Squared:__

Lane 2 (A) 65 degrees Celsius Lane 3 (B) 64.3 degrees Celsius Lane 4 (C) 56 degrees Celsius Lane 5 (D) 55 degrees Celsius

__Analysis:__

Despite that bands appeared at the right height of the 100 base pair ladder, there was too much bright bands at the bottom of the gel. Dr. B. told me to do another PCR Squared gel using the secondary PCR from lane 4 because the PCR Squared in figure 13 will probably have a low yield.



Figure 18. Primary and Secondary PCR of //P. falciparum//. Lane 1 and Lane 10 have the 100 base pair ladder. Lane 2 has the primary PCR and lanes 3-9 have the secondary PCR at different annealing temperatures and times.

__**Primary PCR**__

98 degrees Celsius_ 2 minutes 98 degrees Celsius_ 20 Seconds 58 degrees Celsius_ 20 Seconds ) 72 degrees Celsius_ 20 Seconds Repeat 2-4, 20 cycles 72 degrees Celsius_ 2 minutes 4 degrees Celsius_ Infinite

__**Secondary PCR**__

98 degrees Celsius_ 2 minutes 98 degrees Celsius_ 20 Seconds ? degrees Celsius_ 20 Seconds ANNEALING TEMPERATURE (See Below) 72 degrees Celsius_ 20 Seconds Repeat 2-4, 25 cycles 72 degrees Celsius_ 2 minutes 4 degrees Celsius_ Infinite

Annealing Temperatures__

Lane 3 (A) 65 degrees Celsius Lane 4 (B) 64.3 degrees Celsius Lane 5 (C) 63.1 degrees Celsius Lane 6 (D) 61.3 degrees Celsius Lane 7 (E) 59 degrees Celsius Lane 8 (F) 57.3 degrees Celsius Lane 9 (G) 56 degrees Celsius

Analysis: My target changed from //T. brucei// to //P. falciparum//, but remains Serine thereonine protein phosphatase. The primary came out as a smear at the appropriate height and the secondary in lanes 3, 4, 6 and 9 came out as bright bands at the appropriate height. There is presence of some contamination, but not an incredible amount. I will move on to PCR squared using Lane 3's secondary PCR.

Figure 17. 1st and Last Primer PCR and gel ran on 10/26/2013. Lane 1 and Lane 10 have the 1kb ladder. Lanes 2-9 have the 1st and Last Primer PCR run at different annealing temperatures. (See Below) The Primary from lane 2 in figure 8 was used.

98 degrees Celsius_ 30 Seconds 98 degrees Celsius_ 10 Seconds ? degrees Celsius_ 20 Seconds ANNEALING TEMPERATURE (See Below) 72 degrees Celsius_ 30 Seconds Repeat 1-4, 25 cycles 72 degrees Celsius_ 2 minutes 4 degrees Celsius_ Infinite

__Annealing Temperatures__

Lane 2 (A) 61 degrees Celsius Lane 3 (B) 59.6 degrees Celsius Lane 4 (C) 57.4 degrees Celsius Lane 5 (D) 54.2 degrees Celsius Lane 6 (E) 50.1 degrees Celsius Lane 7 (F) 47 degrees Celsius Lane 8 (G) 44.6 degrees Celsius Lane 9 (H) 43 degrees Celsius

__Analysis:__

After speaking with Dr. B. he told me to run a first and last primer PCR and if it failed, then I should talk to him about switching targets. Since only primer dimers should up in the lanes, and the bands showed up as low smears, it failed.

__**Week 7 & 8**__
 * Great work keeping your data updated on the page. Nice work with the captions and analysis. Where are you with virtual work? Thank you. -Max 10/21/13
 * What I did: **


 * Continue attempting Primary and Secondary PCR at different times and temperatures**
 * Virtual Lab Work**

Figure 16.
 * Secondary PCR and gel ran on 10/9/2013. Lane 1 and Lane 10 have the 1kb ladder. Lanes 2-9 have the Secondary PCR run at different annealing temperatures. (See Below) This Secondary PCR used the Primary from lane 3 in figure 8.**


 * 98 degrees Celsius_ 30 Seconds**
 * 98 degrees Celsius_ 10 Seconds**
 * ? degrees Celsius_ 30 Seconds ANNEALING TEMPERATURE (See Below)**
 * 72 degrees Celsius_ 30 Seconds**
 * Repeat 1-4, 25 cycles**
 * 72 degrees Celsius_ 2 minutes**
 * 4 degrees Celsius_ Infinite**


 * __Annealing Temperatures__**


 * Lane 2 (A) 58 degrees Celsius**
 * Lane 3 (B) 57.8 degrees Celsius**
 * Lane 4 (C) 57.4 degrees Celsius**
 * Lane 5 (D) 56.9 degrees Celsius**
 * Lane 6 (E) 56.2 degrees Celsius**
 * Lane 7 (F) 55.7 degrees Celsius**
 * Lane 8 (G) 55.3 degrees Celsius**
 * Lane 9 (H) 55 degrees Celsius**


 * __Analysis:__**


 * Since this Secondary PCR did not work properly I will probably not use it. I will retry a Secondary PCR at a lower temperature will the Primary PCR from figure 9.**

Figure 15.
 * Secondary PCR and gel ran on 10/9/2013. Lane 1 and Lane 10 have the 1kb ladder. Lanes 2-9 have the Secondary PCR run at different annealing temperatures. (See Below) This Secondary PCR used the Primary PCR from lane 2 in figure 8.**


 * 98 degrees Celsius_ 30 Seconds**
 * 98 degrees Celsius_ 10 Seconds**
 * ? degrees Celsius_ 30 Seconds ANNEALING TEMPERATURE (See Below)**
 * 72 degrees Celsius_ 30 Seconds**
 * Repeat 1-4, 25 cycles**
 * 72 degrees Celsius_ 2 minutes**
 * 4 degrees Celsius_ Infinite**


 * __Annealing Temperatures__**


 * Lane 2 (A) 58 degrees Celsius**
 * Lane 3 (B) 57.8 degrees Celsius**
 * Lane 4 (C) 57.4 degrees Celsius**
 * Lane 5 (D) 56.9 degrees Celsius**
 * Lane 6 (E) 56.2 degrees Celsius**
 * Lane 7 (F) 55.7 degrees Celsius**
 * Lane 8 (G) 55.3 degrees Celsius**
 * Lane 9 (H) 55 degrees Celsius**


 * __Analysis:__**


 * I will redo this Secondary PCR with the same Primary PCR at a lower temperature and see if the amplification increases.**

Figure 14.
 * Primary PCR and Secondary PCR gel ran on 10/8/2013. Lane 1 has the 1kb ladder. Lanes 2-3 have the primary PCR of a different oligo mix from the Secondary PCR. Lanes 4-9 have the Secondary PCR run at different annealing temperatures. (See Below) **


 * 98 degrees Celsius_ 30 Seconds **
 * 98 degrees Celsius_ 10 Seconds **
 * ? degrees Celsius_ 30 Seconds ANNEALING TEMPERATURE (See Below) **
 * 72 degrees Celsius_ 30 Seconds **
 * Repeat 1-4, 25 cycles **
 * 72 degrees Celsius_ 2 minutes **
 * 4 degrees Celsius_ Infinite **


 * __ Annealing Temperatures __**


 * Lane 2 (A) 58 degrees Celsius **
 * Lane 3 (B) degrees Celsius **
 * Lane 4 (C) degrees Celsius **
 * Lane 5 (D) degrees Celsius **
 * Lane 6 (E) degrees Celsius **
 * Lane 7 (F) degrees Celsius **
 * Lane 8 (G) degrees Celsius **
 * Lane 9 (H) degrees Celsius **


 * __ Analysis: __**
 * The Secondary PCRs still came out too low, but the Primary PCR came out as smears and were higher. I will ask Dr. B. about this tomorrow (10/09/13).**



Figure 13.
 * Secondary PCR and gel ran on 10/7/2013. Lane 1 and Lane 10 have the 1kb ladder. Lanes 2-9 have the Secondary PCR run at different annealing temperatures. (See Below)**


 * 98 degrees Celsius_ 30 Seconds**
 * 98 degrees Celsius_ 10 Seconds**
 * ? degrees Celsius_ 30 Seconds ANNEALING TEMPERATURE (See Below)**
 * 72 degrees Celsius_ 30 Seconds**
 * Repeat 1-4, 25 cycles**
 * 72 degrees Celsius_ 2 minutes**
 * 4 degrees Celsius_ Infinite**


 * __Annealing Temperatures__**


 * Lane 2 (A) 58 degrees Celsius**
 * Lane 3 (B) 57.8 degrees Celsius**
 * Lane 4 (C) 57.4 degrees Celsius**
 * Lane 5 (D) 56.9 degrees Celsius**
 * Lane 6 (E) 56.2 degrees Celsius**
 * Lane 7 (F) 55.7 degrees Celsius**
 * Lane 8 (G) 55.3 degrees Celsius**
 * Lane 9 (H) 55 degrees Celsius**


 * __Analysis:__**


 * The secondary PCR band is lower than it should be. It is less than 500 base pairs. The bands that showed up are the result of "primer dimers" and not the result of amplification. This means that the forward and reverse primers are not latching on properly. I was told by Dr. B. to rerun my primary PCR until I get a smear again. I will do this and hopefully get it to work and then rerun my secondary PCR. Eventually I will move on to PCR squared, PCR clean-up and Cloning. Even though this did not amplify, I was happy to at least see some kind of band in secondary PCR. I have been working on this since the summer and have not gotten anything.**

__Week 5 & 6**__


 * Great detailed captions and good analysis. Keep up the nice work. Thank You. -Max 10/07/2013


 * What I did:**


 * First and Last Primer PCR at different times and temperatures**
 * Primary and Secondary PCR at different times and temperatures**
 * Virtual Lab work (not shown, but in progress)**

Figure 12.
 * Lane 1 and 10 have the 1kb ladder. Lanes 2-3 have two different runs of Primary PCR. Lanes 4-9 have Secondary PCR at different annealing temperatures (see below) but did not appear.**


 * (I had cropped both figures 5 and 6 before I uploaded them but they uploaded uncropped for some reason.)**


 * __Primary PCR__**


 * 98 degrees Celsius_ 30 Seconds**
 * 98 degrees Celsius_ 10 Seconds**
 * 58 degrees Celsius_ 30 Seconds**
 * 72 degrees Celsius_ 30 Seconds**
 * Repeat 1-4, 20 cycles**
 * 72 degrees Celsius_ 2 minutes**
 * 4 degrees Celsius_ Infinite**


 * __Secondary PCR__**


 * 98 degrees Celsius_ 30 Seconds**
 * 98 degrees Celsius_ 10 Seconds**
 * ? degrees Celsius_ 30 Seconds ANNEALING TEMPERATURE (See Below)**
 * 72 degrees Celsius_ 30 Seconds**
 * Repeat 1-4, 25 cycles**
 * 72 degrees Celsius_ 2 minutes**
 * 4 degrees Celsius_ Infinite**


 * __Annealing Temperatures__**


 * Lane 4 (A) 82 degrees Celsius**
 * Lane 5 (B) 81.4 degrees Celsius**
 * Lane 6 (C) 80.2 degrees Celsius**
 * Lane 7 (D) 78.3 degrees Celsius**
 * Lane 8 (E) 76 degrees Celsius**
 * Lane 9 (F) 74.4 degrees Celsius**


 * __Analysis:__**


 * I am still not sure why nothing showed. I am especially confounded about the primary PCR. I was sure that would at least show up as a smear, but it did not. I will continue to change the temperature and times for the secondary PCRs until I get it right.**


 * I did some online research on my target and it said that a good temperature is 50 Celsius for the annealing temperature. This is a lot lower than I would have thought, but I will try going to lower temperatures and see if that works. The lower temperatures may work better because it may denature at the higher temperatures. My next run will be on 10/7/2013 for secondary PCR at lower annealing temperatures.**

Figure 11.
 * First and Last Primer PCR. Lane 1 has the 1 kb ladder. Lanes 2-9 have the First and Last Primer PCR at different annealing temperature. (see below).**


 * __Lanes 2-9__**


 * 98 degrees Celsius_ 30 Seconds**
 * 98 degrees Celsius_ 10 Seconds**
 * ? degrees Celsius_ 30 Seconds ANNEALING TEMPERATURE (See Below)**
 * 72 degrees Celsius_ 30 Seconds**
 * Repeat 1-4, 20 cycles**
 * 72 degrees Celsius_ 2 minutes**
 * 4 degrees Celsius_ Infinite**


 * __Annealing Temperatures__**


 * Lane 2 (A) 64 degrees Celsius**
 * Lane 3 (B) 63.3 degrees Celsius**
 * Lane 4 (C) 62 degrees Celsius**
 * Lane 5 (D) 60.2 degrees Celsius**
 * Lane 6 (E) 57.8 degrees Celsius**
 * Lane 7 (F) 56.1 degrees Celsius**
 * Lane 8 (G) 54.7 degrees Celsius**
 * Lane 9 (H) 53.7 degrees Celsius**


 * __Analysis:__**


 * I have decided to just try and rerun secondary PCR. I could not get First and Last Primer to work successfully.**

Figure 10.
 * First and Last Primer PCR. Lane 1 and Lane 10 have the 1 kb ladder. Lanes 2-4 have the First and Last Primer PCR at one temperature and 5-7 have the First and Last Primer at another temperature. (see below).**


 * __Lanes 2-4__**


 * 98 degrees Celsius_ 30 Seconds**
 * 98 degrees Celsius_ 10 Seconds**
 * 58 degrees Celsius_ 30 Seconds**
 * 72 degrees Celsius_ 30 Seconds**
 * 72 degrees Celsius_ 2 minutes**
 * 4 degrees Celsius_ Infinite**


 * __Lanes 5-7__**


 * 98 degrees Celsius_ 30 Seconds**
 * 98 degrees Celsius_ 10 Seconds**
 * 58 degrees Celsius_ 30 Seconds**
 * 72 degrees Celsius_ 30 Seconds**
 * 72 degrees Celsius_ 2 minutes**
 * 4 degrees Celsius_ Infinite**

Week 3 & 4
 * Ramiro - Ok keep attempting the PCR and changing the conditions to get it to work. . Crop your images before uploading - ideally you can crop them in the Imaging Software that is on the UV camera computer. - Dr. B 100113**


 * __ What I did: __**


 * Primary PCR gel**
 * Primary and Secondary PCR together**
 * First and Last Primer PCR ( 1 & 36, 3 & 34)**

__ Figure 9. __
 * First and Last Primer PCR. Lane 1 has the ladder. Lane 2 has 1 and 36 (C2 and F1). Lane 3 has 3 and 34 (C4 and E11). No lanes showed up in lanes 2 and 3. **



__Figure 8.__
 * This gel became a disaster because I dropped it right before I was going to put it in the viewing machine.**
 * Lane 1 has the 1 kb ladder, Lane 2 has the primary PCR, and lane 3 has the secondary PCR. You can not see if there is a result or not since it broke. I will redo this again__.__**

__Figure 7.__
 * __Primary PCR and Secondary PCR and gels round 1__**
 * Lane 1 has the 1 kb ladder and lane two has the primary PCR. The smear came out low and that could be because it wasn't left in there as long as it should have been because I had to leave. I will rerun the primary PCR when I run my secondary PCR.**

__**Week 1 & 2**__


 * What I did:**


 * Pymol Refresher**



** Figure 6A (Left) and 6B (Right). DHFR with the know inhibitor TMQ. It is colored by chains (red, cyan, orange and green lines) with the polar contacts in yellow, TMQ in purple blue sticks, NAP in wheat sticks, and the active site in forest green sticks. **



** Figure 5. The aligned human DHFR (1U72) and the potential target (3CL9). After alignment the RMS value was found to be: 1.126 (111 to 111 atoms). 1U72 is shown as green carbons and orange sticks for its active site, while 3CL9 is shown as cyan carbons and magenta sticks for its active site. The MTX are the corresponding colors shown as sticks. **



** Figure 4. Human DHFR with MTX. NDP is the yellow sticks, MTX is the magenta sticks, the active site is the orange sticks nd the rest of Human DHFR is colored by element with green carbon lines. **

** Figure 3 The BLAST report for 1U72 with 3CL9. **



** Figure 2. The A chain of DHFR with the know inhibitor MTX (magenta sticks). In addition, the polar contacts are in yellow lines, the active site is orange and the rest is colored by element with carbon as green. **

**Figure 1. The natural substrates for DHFR-TS with the top chain in green, bottom chain in red, the polar contacts are yellow, the hydrophobic residues are blue sticks, the ionic residues are magenta sticks, and the polar residues are orange sticks. **


 * __Analysis: __**


 * I looked at several proteins on PyMol (2H2Q, 1U72, 3CL9, 3HBB) and organized their different sections in so they could be better distinguished. I separated the chains of proteins by color and the active site and substrates by color and showed them as sticks. I aligned 1U72 and 3CL9 to show the similarities among them. 1U72 and 3CL9 are very similar with only a few different amino acid groups (could account for the slight difference in the position of MTX in the active sites). In addition I did a BLAST for 1U72 and 3CL9 (Figure 3). **


 * As far as errors there could have been some computer errors in the colors. By this I mean that come colors might not be what they say they represent in the captions. Although this is unlikely it is a possible form of error. **


 * __Conclusion: __**


 * The PyMol refresher was very helpful because I had forgot so many commands and things that were basic in the second half of the spring semester. For instance I had to ask how to find the RMS value when I aligned 1U72 and 3CL9 and afterwards I remembered how it automatically shows up after you put align from the A tab. Overall this refresher will helped me to remember some of the basic functions and techniques of PyMol as well as the websites (PDB, BLAST) that we can use. These procedures will be useful in future Virtual Screening and PyMol labs that are done this semester in the VDS lab and in other labs. **
 * Fall Semester 2013 above**


 * __SUMMER__**
 * Week 8**



Figure 21
 * __Overlap PCR round 4 with Secondary overlap in white color. The secondary overlap should have shown up to the right of the ladder, but did not.__**



Figure 20
 * __Overlap PCR round 3 with Secondary overlap in white color. The secondary overlap should have shown up to the right of the ladder, but did not.__**

Week 7 Figure 19: Figure 18:
 * __Overlap PCR round 2 with Secondary overlap in white color. The secondary overlap should have shown up to the right of the ladder, but did not. The ladder for some reason showed up as a smear.__**
 * __Overlap PCR with Primary and Secondary overlap in red color. The primary overlap is shown as a smear next to the ladder and the secondary overlap should have shown up to the right of the primary overlap, but did not.__**

Figure 17:
 * __Overlap PCR with Primary and Secondary overlap in non-color. The primary overlap is shown as a smear next to the ladder and the secondary overlap should have shown up to the right of the primary overlap, but did not.__**

Week 6
 * __Ramiro - show your Overlap PCR results and secondary PCR - Dr. B 071713__**

Pfxdr spin down, Pflc Gel Filtration for Pfxdr, VS4 redo redo,

Week 5

RE Digest, Protein Characterization of Pfxdr Figure 16.
 * __ PCR gel of Pfdxr before drying. Elution 2 (Bottom), Elution 1, Wash, Flow Through, Soluble Fraction, Cell lysate After Induction, Cell Lysate Before Induction, and Ladder (Top). __**

Figure 15. Figure 14.
 * __Dried PCR gel of Pfdxr. Elution 2 (Left), Elution 1, Wash, Flow Through, Soluble Fraction, Cell lysate After Induction, Cell Lysate Before Induction, and Ladder (Right).__**
 * __Dried PCR gel of Pfdxr. Elution 2 (Top), Elution 1, Wash, Flow Through, Soluble Fraction, Cell lysate After Induction, Cell Lysate Before Induction, and Ladder (Bottom).__**

Figure 13.
 * __Dried PCR gel of Pfdxr. Elution 2 (Left), Elution 1, Wash, Flow Through, Soluble Fraction, Cell lysate After Induction, Cell Lysate Before Induction, and Ladder (Right).__**

Week 4

Protein Purification, Protein Characterization, Homemade SDS-Page Gel Solutions Figure 12.
 * __Elution 2 Nanodrop from Protein Purification__**

Figure 11.
 * __Elution 1 Nanodrop from Protein Purification__**

Week 3

PCR for RE Cloning of pGFP (Green), Midi Prep Kit.

Figure 10.
 * __PCR gel image for pGFP (Green protein).__**

Week 2

PCR for PLGB22 (Purple), Nanodrop and DNA Sequencing for that plasmid.


 * __ NNNNNNNNNNCNGCNNTCGACTCTAGANNNNNCCGGGTACCGAGCTCGAATTCACTGGCCGTCGTTTTACAACGTCGTGA __**
 * __ CTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGG __**
 * __ CCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTTACGCAT __**
 * __ CTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACA __**
 * __ CCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCG __**
 * __ GGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTA __**
 * __ TAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTG __**
 * __ TTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAA __**
 * __ GGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCAC __**
 * __ CCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAG __**
 * __ CGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGG __**
 * __ TATTATCCCGTATTGACGCCGGGCAAGANCAACTCGGTCNCNNCATACNCTATTCTCNNNANGACTNNGNTNNANTACTN __**
 * __ NNCAGTCNCNNAAAANCNTCTTANGNATGGCATGACNNNANANNNNATGCANNGCTGCCATNANNNNNANTGANANNNNG __**
 * __ NNNNNTTANNNNGANNNNATNNNANNNCNAGNANNNANNNTTTTTNCNNNNNNNGGGGGNTNNNGTANNNNNNNNNTNNN __**
 * __ NNNNNNNNNNANNANNNNNNNNNNNNNNNNNNNNNNNNCNNNNNNNNNNNNNANNNNNNNNNNNNNNNNNNNNNNNNANN __**
 * __ NNNNNNNNNNNNNNNNNNNNNNNNNNNNN __**

Figure 9.
 * __DNA Sequencing results for Order 91977. __**



Figure 8.
 * __PCR for PLGB22 (Purple). My ladder is the one on the right and my B tube is shown with a brighter band.__**

Figure 7.
 * __Nanodrop with a 260/280 of 1.88, a 260/230 of 2.27 and ng/ul of 88.3.__**

Figure 6.
 * __ Nanodrop with a 260/280 of 1.86, a 260/230 of 2.26 and ng/ul of 89.0. __**

Week 1

Quantifying DNA using Nanodrop,Submitting DNA to DNA Sequencing Facility, Made LB media and LB media plates + AMP for Puc19, Transformation of competent cells for plasmid prep

Figure 5.
 * __ Nanodrop with a 260/280 of 1.79, a 260/230 of 2.20 and ng/ul of 3075.5. __**

Figure 4.
 * __ Nanodrop with a 260/280 of 1.73, a 260/230 of 2.20 and ng/ul of 3844.4. __**

Figure 3.
 * __Plate A of DHS Alpha plasmid with LB media + AMP.__**

Figure 2.
 * __ Plate B of DHS Alpha plasmid with LB media + AMP. __**

Figure 1.__** Plate C of DHS Alpha plasmid with LB media + AMP.