Saniya+H.+(Springer)

__**Weeks 14&15**__



Analysis: PCR clean up results done with Stacy can be seen in Figure 1. After using the Sigma-Aldrich PCR Clean up kit, Nanodrop spectrophotometer was used to measure the absorbance and concentration of the PCR squared sample. A yield of 160.5 ng/uL was found. The 260/280 ratio was close to 1.80, expressing the sample's purity. 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.

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



Analysis: Figure 1 above shows significant smearing and contamination in the PCR2 sample. Two strong bands along with the smear can be seen in lanes 5 and 6 while lanes 3 and 4 show faint bands. Distinct bands can be seen near 735 bp which is the size of my gene of interested but the other band lies around 200-300 base pairs which could signify a potential primer dimerization. These floating pieces of DNA may or 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.

1162014- Include more pictures __**Weeks 8&9&10**__



Analysis: The gel above is the gel for the second run of PCR squared using the same PCR squared product from the first run. The first run done showed nothing on the gel and thus we thought there might have been something wrong with the gel and decided to run the sample again. However even on the second run there was only a slight improvement. It is hard to see it in the image above but there were light bands, in then right area, that showed up on the first 3 lanes of sample which were my samples of PCR squared. Although this means there is something there - it does not match up because as can be seen below, secondary PCR resulted in a bright and concise band. PCR squared should be a way to affirm those results rather than show a problem now. So we just did the whole PCR reaction again using the secondary PCR product and will run the gel in the coming week and hope to see a change in the results.

9232014- Nice work, keep it up __**Weeks 5&6&7**__

10/10/14: Secondary PCR



Analysis: My lane, lane 3, showed a band with some smearing meaning my secondary PCR was a success. The band is located close to the area of 750 bp which is a good place because my gene of interest is at 735 bp.

Primary PCR

Trial 2 --> 10/03/14



Trial 1 --> 10/02/14



Analysis: The first gel run for primary PCR did not show anything. This might have been because of the temperatures and time durations used for the PCR machine. This is why for the second trial of primary PCR, the NEB recommended guidelines for Q5 polymerase were used instead. A DNA smear was the result of this change and is shown in Trial 2's gel image!

10/01/14: Plasmid Midi-Prep





Analysis: Midi Prep was done to isolate the plasmid, pNIC-BSa4, out of the pellet and away from the rest of the unwanted cell components. The concentration of DNA found at the end of the protocol was very high which could be attributed to too much pellet being used but it is still odd because this plasmid is a low-copy plasmid.

9/30/14: PCR Primer Design Tails for pNic28-Bsa4 Cloning

Forward Primer: 5’ TACTTCCAATCCATGATGGAAATCTCTCTGCTGAC 3’

Reverse Primer: 5’ TATCCACCTTTACTGTTAAACAGCTTCAGATTCAA 3’





Analysis: A forward and reverse primer were designed for PCR to amplify the CDS of my gene of interest to synthesize Compatible Ends suitable for Ligation Independent Cloning (LIC) for insertion into the pNIC-Bsa4 as the accepting vector for eventual expression. A virtual circular sequence was created from NEB cutter to represent pNIC28-Bsa4 plasmid with BsaI cuts. BsaI was removed in order to allow the insertion of the CDS of STP1 for cloning. On the pNIC28-Bsa4 plasmid sequence, BsaI was removed and the CDS of MtPtpA was inserted instead for cloning purpose.

__**Week 3&4**__

PCR:

Analysis: The purpose of this lab was to amplify purple protein coding sequence. Dilutions were made for the tubes - t ube A had 1:10000, tube B had 1:1000, and tube C had 1:100. After PCR, gels were made to analyze the samples to see if the DNA bands would be present. The gel was run for about 44 mins, at 105 volts until the visible blue marker dye was near the bottom 1/4th of the gel. For lanes 2-5, my samples, DNA bands showed where they were supposed to!

RE Digest:



Analysis: RE Digest was done to make cuts in the plasmid to have optimal gene insertion. EcoRI, PvuII, and a combination of both were used in unique samples of the plasmid. Running the samples in the gel resulted in the correct number of bands in each lane - 1 for the one cutter EcoRI, 3 for both the one cutter EcoRI and two cutter PvuII, and 2 for the two cutter PvuII. There seems like there could be a little bit of contamination but most likely not.

Day 3: Transformation





Day 2: Transformation



Analysis: Days 2 and 3 of Transformation of bacterial colonies with the plasmid pNIC-Bsa4 in competent E. coli cells was performed. This was successful - the pellets can be seen in the figures under Day 3. No contamination seems to be present. They were stored for later use for Midi Prep.

Re Do Transformation:





Analysis: As seen in the two images above, the transformation was not successful even on the second round of performing it. There was no growth of bacteria present. There may have been one small colony in the 10 ul sample but that would not be a viable option for further experiment. The lack of growth may have been because the DHFalpha cells were the same as the ones used on the first try for transformation and thus there is something wrong with those cells specifically.



982014- Good job, and next time you know what to do for a viable option for further experiment


 * __Week 1&2__**





Analysis: As seen in the two images above, the transformation was not successful even on the second round of performing it. There was no growth of bacteria present. There may have been one small colony in the 10 ul sample but that would not be a viable option for further experiment. The lack of growth may have been because the DHFalpha cells were the same as the ones used on the first try for transformation and thus there is something wrong with those cells specifically. not used within an appropriate time after being removed from the freezer and thus they may have died or become incompetent for the transformation to be successful.





Analysis: The purpose of doing this lab was to use the Nanodrop to determine the concentration of pgbr22. Unfortunately our graphs were put on the wrong setting and thus we were not able to acquire the correct concentration as is seen with our units in mg/ml instead of ng/ul. However, the peak of absorbance is still at 260 nm for both graphs which is where the absorbance should be for a protein. The concentration written on the tube of 238.4 ng/ul was used to calculate the volume of plasmid needed for the mixture to send to DNA sequencing.