Stacy+T.+(Springer)

__**Weeks** 14 & 15__
__Good work Stacy - Dr. B 1211214__

Analysis:
In this cloning procedure, the accepting vector, pNIC-Bsa4 plasmid, must be prepared and cut using the appropriate restriction endonuclease - in this case, BsaI. The buffer used for BsaI-HF is the NEB Cutsmart buffer according to the NEB website. After the plasmid was cut, the samples are combined and cleaned up using PCR Clean-Up kit from Sigma-Aldrich. Instead of eluting with 50 uL of elution buffer, only 30 uL was used to prevent excess loss of the sample. Figure 1 shows the nanodropped concentration of the clean up sample. The concentration is that of 27.4 ng/uL, a big loss of concentration compared to the uncut pNIC-Bsa4 concentration of about 358 ng/uL. This is due to the cutting of the SacB gene (~2000 bp) out of the plasmid (pNIC-Bsa4 has ~7000bp). Some possible errors that may have occurred include incubating the samples in the hot water bath of 37 degrees Celsius for a bit less than 2 hours, not allowing the restriction enzyme to cut enough of the plasmid accurately.

__**Weeks** 11,12 &13__






__Analysis:__ Secondary PCR temperature gradient was conducted from 55-66.5 degrees Celsius due to the multiple failed rounds of secondary PCR using the NEB guidelines and annealing temperature of 58 degrees Celsius. All of the temperatures resulted in strong bands except lane 9's sample with an annealing temperature of 55 degrees Celsius. Lane 4 (64.3 degrees Celsius annealing temperature) had the strongest band and all of the bands are located between the 500 to 1000 base pairs band on the 1kb ladder. (My gene is 738 base pairs long). There is slight contamination in all of the samples shown as smears on the gel. Because the secondary PCR temperature gradient resulted in strong band at 64.3 degrees Celsius, this sample was ran through PCR squared to further amplify the amount of DNA in greater amounts since PCR clean up will result in a loss of significant amounts of sample. Figure 4 shows the results from PCR squared at 64.3 degrees Celsius. There are significant smearing and contamination in the PCR2 sample and faint bands can be seen below the 500 base pairs mark on the 1 kb ladder in lane 2. The faint bands around 200-300 base pairs signifiy several potential primer dimerization. These floating DNA pieces may not necessarily be cleaned up in PCR clean up but will not affect cloning later as they do not have cohesive ends to stick to the plasmid. PCR clean up results can be seen in FIgures 5 and 6. After using the Sigma-Aldrich PCR Clean up kit and following the procedures, nanodrop spectrophotometer was used to measure the absorbance and concentration of the PCR squared sample. Figure 5 shows a yield of 160.5 ng/uL and figure 6 158.1 ng/uL, both great significant concentration yields of the combined PCR squared samples. The 260/280 ratios are also close to 1.80 which signifies the sample's purity. Now that the gene of interest has been successfully synthesized using PCR, the sample can be used for cloning. Next steps in this research study would be pNIC-Bsa4 plasmid preparation and cohesive end generation for the purpose of cloning the gene into low-copy-plasmid pNIC-Bsa4. PCR is crucial in any research study that requires the synthesis of new DNA with a specific gene of interest.

1162014- Where is week 10? Great job with the rest however
 * 12042014- Where is week 11,12,&13?**
 * Dr. B, I did weeks 8,9, and 10 together but I just forgot to put "& 10" on the headline.

__**Weeks** 8,9 &10__
** PCR Squared **

** Secondary PCR trial 2 **

__Analysis:__ Secondary PCR trial was conducted again to obtain a more visible band. As shown in Figure 2, a light band between 500-1000bp can be seen on lane 3. This faint band may be due to a lack of concentrated Taq polymerase used during the procedure. A PCR squared was ran using this sample while another secondary PCR was ran again on the same gel as shown in Figure 1 to ensure that the gene of interest has been synthesized in a full length DNA strand and amplified properly. Figure 1 shows almost no bands at all from the PCR squared. A new PCR squared will be ran and another secondary PCR sample will be made using temperature gradient to obtain a more visible band.

10232014- Job Well Done

__**Weeks** 5,6,&7__
__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. Figure 3 shows an unsuccessful secondary PCR result for Jairo (lane 5) and myself (lane 3) since no DNA bands corresponding to the length of our coding DNA sequence appeared on the agarose gel. Saniya's secondary PCR results worked successfully, allowing me to believe that the absence of a DNA band from my sample may have simply been due to a human error in pipetting not enough of the Q5 hotstart polymerase. The DNA band (Saniya's) that appeared on the gel seems to correspond to a DNA length of about 700-800 base pairs when compared to the 1kb DNA ladder. This is a positive outcome since all three of us have the same target protein that consists of 738 base pairs. Secondary PCR will be conducted again this coming week to achieve a strong DNA band on the agarose gel via gel electrophoresis.
 * Secondary PCR **


 * Plasmid pNIC-Bsa4 midiprep **

__ 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 260nm wavelength to confirm the presence of only pNIC-Bsa4 and determine the concentration of the plasmid. Figure 1 and 2's (above) concentrations show an average of about 347.2 ng/uL of pNIC-Bsa4. Although the graphs show consistent line and a peak absorbance at 260nm, the high concentration of midi-prep results is uncommon since pNIC-Bsa4 is a low copy plasmid that should present a concentration of about 60-100 ng/uL. This excess concentration may be due to a high amount of bacterial pellets being midiprepped or the presence of chromosomal DNA that didn't get filtered out. Future uses of midiprep results may be slightly affected during cloning as the plasmid concentration will have to be diluted so as to have a lower plasmid to gene ratio. Midiprep is a crucial process in discovering novel inhibitors for my target SaSTP as the process isolates pNIC-Bsa4 plasmid (which will be used in RE digest and cloning with desired gene insert) from the rest of the DH5-alpha bacterial pellets.

__ 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 with Saniya and Gio failed as there was no presence of a smear at all on the agarose gel as seen in Figure 1 above. 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. As shown on Figure 1 above, 6 samples of primary PCR from different people were ran on the same gel but alas, none of the samples worked/showed any smearing. This issue was remedied by changing the thermocycler cycling conditions and adjusting the annealing times so as to correspond to the NEB recommended guidelines for Q5 polymerase. After modifying the conditions of the PCR machine, a smear was visible on both mine and Saniya's samples, as shown in Figure 2 above. My primary PCR sample on lane 3 of Figure 2 shows a strong/darker smear, signifying a greater number of DNA strands of that length in the sample.
 * Primary PCR **


 * Primer tail design of F&R primers of STP1 **





__Analysis:__ Forward and reverse primers were designed to be tailored to the //Streptococcus agalactiae// serine threonine phosphatase gene. Forward and reverse primers are needed to properly conduct primary PCR and make multiple copies of the STP1 gene with sticky ends. The primers function to allow the polymerase to bind to the correct area of the gene and start replication. In this way, sticky ends of the gene are also created to make annealing to vector easier during cloning.

09232014- Great Job

**Primary PCR of pGBR22 and M13F&R primers**
Lane 1: 100bp marker Lane 2: Tube A - Saniya (0.3ng of template DNA pGBR22) Lane 3: Tube B - Saniya (3ng of template DNA pGBR22) Lane 4: Tube C - Saniya (30ng of template DNA pGBR22) Lane 5: Tube D - Saniya (no template DNA) Lane 6: Tube A - Stacy (0.3ng of template DNA pGBR22) Lane 7: Tube B - Stacy (3ng of template DNA pGBR22) Lane 8: Tube C - Stacy (30ng of template DNA pGBR22) Lane 9: Tube D - Stacy (no template DNA)

__**Analysis:**__ My first primary PCR was almost successful except for the absence of a DNA band on Lane 7 (Tube B). This may have been due to the lack of concentration of Taq DNA Polymerase or not putting enough blue juice into sample tube B. Tube D (lanes 5 and 9) did not have a DNA band due to the absence of template DNA of pGBR22 in the sample as this tube served as a control for the procedure.

Restriction Enzyme Digest
Lane 1: skipped Lane 2: 1kb DNA ladder Lane 3: uncut plasmid Lane 4: pGBR22 + EcoRI (Saniya) Lane 5: pGBR22 + EcoRI+PvuII (Saniya) Lane 6: pGBR22 + PvuII (Saniya) Lane 7: pGBR22 + EcoRI (Stacy) Lane 8: pGBR22 + EcoRI+PvuII (Stacy) Lane 9: pGBR22 + PvuII (Stacy)

__**Analysis:**__
Results of the restriction enzyme digest of pGBR22 show a successful, completed digestion of pGBR22 by EcoRI and PvuII restriction enzymes. Since EcoRI is a 1-cutter restriction enzyme, one DNA band is shown on the agarose gel as the plasmid is cut from a circular piece of DNA into one linear strand. PvuII is a 2-cutter enzyme, resulting in 2 DNA bands on the gel and when PvuII and EcoRI are combined, 3 bands are shown on the gel. The uncut plasmid did not show up at all on the gel possibly due to the lack of uncut plasmid pipetted into lane 3 or a small concentration of the plasmid, resulting in the plasmid to not appear on the gel as a band of DNA.

**Day 2&3 Transformation**




__**Analysis:**__ Days 2 and 3 transformation involved growing the transformed DH5-alpha //E. coli// cells in larger quantity with LB media and Kanamycin. Kanamycin was used to prevent growth of other microbes in the LB during incubation period in the shaker. Pellets were then collected via centrifugation and stored in -20 degrees Celsius fridge for purification purposes in the future.

9082014- Keep up the good work

**Bacterial Transformation of pNIC-Bsa4 //E. coli// DH-5 alpha cells**

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


 * Figure 2: ** 10 uL sample of 50 ng of pNIC-Bsa4 plasmid transformed into DH5-alpha // E. coli // cells plated on LB+KAN with SOC media after being incubated overnight in 37 degrees Celsius incubator.

As can be seen from the colonies (dots) in figures 2 and 3, bacterial growth of the DH-5 alpha cells is present on both the 10uL and 50uL agar plates. Since the plates were made from LB and given the antibiotic Kanamycin, only bacteria that has taken up the pNIC-Bsa4 plasmid is able to grow as pNIC-Bsa4 is resistant to Kanamycin. The Kanamycin also acts as a way to prevent contamination and growth of other microorganisms on the plate that may interfere with the transformation of the pNIC-Bsa4 plasmid into the DH-5 alpha cells.
 * __Analysis:__ **

**Nanodrop**

 * Figure 1: ** Nanodrop measurements #1 and 2 of pGBR22 plasmid showing maximum absorbance at a wavelength of 260nm

Nanodrop is a technique used to determine the concentration of a plasmid at maximum absorbance. DNA is absorbed at a maximum wavelength of 260nm and results from the measurements of pGBR22 plasmid confirm that the nanodropped specimen is DNA. The results of the concentrations for trial 1 is that of 257.6 ng/uL and trial 2 for 263.6 ng/uL. Values of the 260/280 ratio include 1.89 and 260/230 as 2.50 and 2.51, indicating purity of the pGBR22 sample. After the concentration of the plasmid has been determined, the plasmid is then sent to DNA sequencing and later analyzed in the computer lab to confirm the validity of the DNA sequence of the pGBR22 plasmid.
 * __Analysis:__ **