Jazmine+A.+(Springer)

__**Week 14 & 15**__
__A nalysis __ I decided to do my oligo mix again because primary PCR was not working and the oligo mix was already two months old. Making my oligo mix again helped because a smear showed up as you can see in Figure 13. Again, this worked because the oligo mix was probably too old making it degraded. The smear is not that distinct because there wasn't that much volume in the well. The smear shows that the oligo primers have overlapped, resulting in a continuous chain of different sized gene sequences.As overlap occurred, this sampcan now undergo secondary PCR to obtain the complete gene sequence for PtpA. No changes were made temperature wise. As shown in Figure 14, there is a distinct band in Lane 5. Primary PCR was not run in this gel due to having a limited amount of it. The reasoning behind why secondary PCR worked is because the sample was mixed with the custom tail primers. The band should be in 500 bp size, but since a 1kb ladder was used, it’s hard to tell. To be noted, Lane 5 has a fainter band due to a far less volume of sample inserted into the well (9 uL) After the complete sequence is obtained, amplification of the amount of PtpA gene is required for clean-up and later cloning. PCR squared was done after secondary PCR. As shown in Figure 15, there are 4 light but distinct bands in Lanes 2 to 4. After PCR squared and PCR clean-up of that reaction, Nanodrop was performed on the purified solution of the mtPtpA gene sequence. The resulting concentration is about 200ng/uL, as shown in Figure 16. Once verification of the purity and concentration of DNA sample are found and determined to be good, the gene can be inserted into a cut plasmid pNIC-Bsa4 through Ligation Independent Cloning.

Figure 16. PCR cleanup Nanodrop check. Concentration was 200.4 ng/ul.

Figure 15: PCR Squared agarose gel Lane 1: 1bp ladder Ladders 2-5: 5µl of PCR Squared Sample



Figure 14: Secondary PCR agarose gel Lane 1: Blank Lane 2-5: PCR squared from other group Lane 6: Secondary PCR

Figure 13:Agarose gel of primary PCR for mtPtpA Lane 1- Nothing  Lane 2- 1kb DNA ladder  Lane 3- Oligo primer mix sample


 * 1242014- Need more pictures and more in depth analysis and captions**

1162014- Include more pictures

9232013- Good job, but you might want to be more descriptive, and include more pictures

__**Week 11, 12, & 13**__
Figure 12: Agarose gel results for Primary and Secondary PCR Lane 1: Empty Lane 2: 1 kb Ladder Lane 3: Primary PCR Lane 4: Secondary PCR

__ Analysis: __ Secondary PCR was conducted with the forward and reverse primers to amplify the full length gene from the pool of DNA fragments in the primary PCR reaction. I continued with Secondary PCR as I thought there was a smear for Primary PCR. However, Secondary PCR did not work, meaning Primary PCR didn't work as well. The presence of a smear on the gel (via gel electrophoresis) signifies the complete synthesis of the full length DNA along with the target gene.

Figure 12: Agarose gel results for Primary and Secondary PCR Lane 1: Empty Lane 2: 1 kb Ladder Lane 3: Primary PCR Lane 4: Secondary PCR

__ Analysis: __ Secondary PCR was conducted with the forward and reverse primers to amplify the full length gene from the pool of DNA fragments in the primary PCR reaction. I continued with Secondary PCR as I thought there was a smear for Primary PCR. However, Secondary PCR did not work, meaning Primary PCR didn't work as well. The presence of a smear on the gel (via gel electrophoresis) signifies the complete synthesis of the full length DNA along with the target gene. Also, I realized after I finished that I had gotten the wrong Primary and Secondary PCR, meaning there is still a possibility that it can work.

__**Week 8, 9, & 10**__
Figure 13: Agarose gel results of Secondary and Primary PCR for Lauren and Jazmine Lane 1: Empty Lane 2: 1 kb ladder Lane 3: Lauren Secondary PCR Lane 4: Jazmine Primary PCR

__ Analysis: __ Primary PCR was conducted to synthesize a full length DNA containing the gene of interest by filling in the gaps between the oligos with dNTPs. The second primary PCR trial I conducted was successful as there is a presence of a smear on the agarose gel as seen in Figure 12. The presence of a smear on the gel (via gel electrophoresis) signifies the complete synthesis of the full length DNA along with the target gene.

__**Weeks** 5,6,&7__

 * Primary PCR **

Figure 11: Agarose gel results of Primary PCR for Lauren, Jazmine, and Alberto. Lane 1: Empty Lane 2: 1 kb ladder Lane 3: Lauren (first failed) Primary PCR Lane 4: Lauren (second attempt) Primary PCR Lane 5: Lauren (second attempt) Primary PCR Lane 6: Jazmine Primary PCR Lane 7: Jazmine Secondary PCR Lane 8: Alberto Primary PCR Lane 9: Alberto Secondary PCR Lane 10: Empty

__ Analysis: __ Primary PCR was conducted to synthesize a full length DNA containing the gene of interest by filling in the gaps between the oligos with dNTPs. The first primary PCR trial I conducted failed as there was no presence of a smear at all on the agarose gel as seen in Figure 11. The presence of a smear on the gel (via gel electrophoresis) signifies the complete synthesis of the full length DNA along with the target gene. I did secondary PCR with my first, but a band did not show up. That means that Primary PCR did not work because Secondary PCR didn't work either.

** Plasmid pNIC-Bsa4 midiprep ** Figure 10: Trial 2 of 2uL sample of pNIC-Bsa4 with absorbance reading of -0.002 at 231nm wavelength. Peaking at 3.7 ng/ul.


 * Figure 9**: Trial 1 of 2uL sample of pNIC-Bsa4 with absorbance reading of 0.009 at 230nm wavelength. Peaking at 4.3 ng/ul.

__ Analysis: __ Plasmid pNIC-Bsa4 Midi Prep was conducted using a Qiagen Midi Prep kit in order to extract the pNIC-Bsa4 plasmid from the rest of the DH5-alpha bacterial pellet and to isolate the plasmid from other cellular structures such as chromosomal DNA, protein, and RNA. Results from Midiprep was nanodropped at 230nm wavelength to confirm the presence of only pNIC-Bsa4 and determine the concentration of the plasmid. Figures 9 and 10 show an average of 4.0 ng/uL of pNIC-Bsa4. This is a very low concentration for pNIC-Bsa4. This may have occurred due to contamination throughout the experiment.

** Primary PCR of pGBR22 and M13F&R primers **
__**Analysis:**__ Overall, my first primary PCR was successful except for one band, which had Tube A. This may have been due to the lack of concentration of Taq DNA Polymerase or taking too long to but the Taq. Tube D, lane 5, did not have a DNA band because it did not have template DNA of pGBR22 in the sample. This was because it was the control in our experiment.


 * Figure 8**: 1% Agarose gel of primary PCR results containing different concentrations of pGBR22, M13 Forward and Reverse primers, dNTPs, buffer, Taq DNA polymerase, blue juice, and nanopure water. A 100bp ladder was placed in lane 1 as a reference point of DNA length.

Lane 1: 100bp marker Lane 2: Tube A (0.3ng of template DNA pGBR22) Lane 3: Tube B (3ng of template DNA pGBR22) Lane 4: Tube C (30ng of template DNA pGBR22) Lane 5: Tube D (no template DNA) Lane 6: Keenan (uncut plasmid) Lane 7: Keenan EcoRII + PvuII Lane 8: Keenan EciRII Lane 9: Keenan PvuII

__**Analysis:**__
The results of the RE digest of pGBR2 are quite different from the results that should have been. My lane 4 is PvuII, which is supposed to have two bands because it is a two cutter. However, my lane 4 only has one band. That means that PvuII didn't recognize the cut that was supposed to be and only recognized one. This made lane 2 only have two bands.


 * Figure 7**: 1% Agarose gel containing 2 samples of restriction enzyme digest from pGBR22 plasmid with EcoRI+PvuII, EcoRI, and PvuII restriction enzymes. A 1kb DNA ladder is placed in Lane 1 as a reference point of DNA length/base pairs

Lane 1: 1kb DNA ladder Lane 2: pGBR22 + EcoRI+PvuII Lane 3: pGBR22 + EcoRI Lane 4: pGBR22 + PvuII


 * Figure 6**: Pellet of pNIC-Bsa4 DH5-alpha transformed E. coli cells after centrifugation at 6000rpm for 15 minutes at 4 degrees Celsius


 * Figure 5**: pNIC-Bsa4 DH-5 alpha cells grown in 80mL of LB media and 80 uL of Kanamycin after incubation in the shaker at 200rpm for 16 hours.

** Bacterial Transformation of pNIC-Bsa4 //E. coli// DH-5 alpha cells **
The colony in figure 4 is shown by a whitish dot, which is bacterial growth of the DH-5 alpha cells is present on the 10uL agar plates. I do not have a 50 uL agar plate with colonies shown because bacteria did not grow. The plates were made from LB and given the antibiotic Kanamycin, which only bacteria that has taken up the pNIC-Bsa4 plasmid is able to grow as pNIC-Bsa4 is resistant to Kanamycin.
 * __Analysis:__ **

*50 uL sample was not pictured due to having no colonies grown.
 * Figure 4: ** 10 uL sample of 50 ng of pNIC-Bsa4 plasmid transformed into DH5-alpha // E. coli //competent cells plated on LB+KAN with SOC media after being incubated overnight in 37 degrees Celsius incubator


 * Figure 3: ** 50 and 10 uL sample of 50 ng of pNIC-Bsa4 plasmid transformed into DH5-alpha // E. coli //competent cells plated on LB+KAN with SOC media before being incubated overnight in 37 degrees Celsius incubator

__ **Analysis:** __ Figure 1 and 2 illustrate peaks at 260nm which is the maximum wavelength DNA absorbs light. The ratio 225/280 in both figures indicates 1.90 and 1.88. The ration 230/280 in both figures indicates 2.47 and 2.42. This concludes that it indicates the purity of the pGBR22 sample. After the concentration of the plasmid is determined, it is sent to DNA sequencing to verify the DNA sequence of the pGBR22 plasmid.




 * Figure 2**: Trial 2 of 2uL sample of pGBR22 with absorbance reading of 2.065 at 230nm wavelength. Peaking at 249.5nm.


 * Figure 1**: Trial 1 of 2uL sample of pGBR22 with absorbance reading of 1.821 at 230nm wavelength. Peaking at 224.6nm.