Katherine+V.

More pNIC-Bsa4 was cut and ran on a gel to ensure the plasmid was indeed cut. Afterwards, another attempt at cloning was performed using the PCR squared reagents previously purified. The plates were left to incubate overnight and will be checked for bacterial growth tomorrow. To attempt cloning once more, more secondary PCR and PCR squared was needed. Two tubes of secondary PCR was created and ran on a 1% agarose gel with a 100bp ladder. Four PCR squared samples were made using both of the secondary PCR's. The gel after PCR squared showed contamination in the 2nd secondary PCR sample, so only the 1st secondary PCR was used for clean up and cloning. __Nice work on the captions and analysis. Did you submit anything to sequencing? Also where are you with virtual work? Thank you. -Max 10/21/13__ __Week of 10/7/13 - 10/11/13__ __Week of 9/30/13 - 10/5/13__** Using the pNIC-Bsa4 cut from last week, cloning transformation was done. But after 2 days of incubation, no colonies grew. All of the times in the transformation protocol were doubled. That could have been a reason why the colonies did not grow; the cells were heat shocked for too long.Tube A plate had 2 uL of accepting vector and 4 uL of insert while Tube B plate had 1 uL of accepting vector and 9 uL of insert. The results of gel extraction was nanopured to yield a high concentration of 451.4 ng/ul. PNIC-Bsa4 was cut with restrictive enzymes for the cloning procedures. Week 3 & 4 9-Sep - 22-Sep PCR squared was made using the secondary PCR from the previous week. Then PCR squared clean up was performed on 4 samples, but yielded very low nanodrop results due to a mistake that occured during the clean up process. Nanopure was added instead of the binding solution, so the DNA was not collected in the filter. The PCR squared protocol was doubled to create 8 samples. The samples were ran again and then used for gel extraction in order to gather a larger amount of DNA. The next steps would include using the gel extraction for cloning. Since the concentration of the PCR squared clean up was too small, gel extraction was performed. There was no ladder to compare the sample with, but all of the lanes seem to have DNA with the same amount of base pairs. The gel was cut and underwent extraction. __Week 1 & 2**__ The DNA sequencing results from the pNIC-Bsa4 cloning were analyzed and blasted. The highest cloning result was 8% coverage and 100% identity. Since the results were very low and had no signs of the inserted vector, a new oligo mix was created using pfSTPP primers. A homology model was started using the SWISS model workspace and a PyMol refresher was completed as well. = =
 * __Week of 10/28/13 - 11/1/13__**
 * __Week of 10/21/13 - 10/25/13__**
 * __Week of 10/14/13 - 10/18/13__**
 * Transformation was attempted again using the recently cut pNIC-Bsa4 and gel extraction product since no colonies grew on the last plate. This time, colonies did grow after two days of incubation in the 37 degree incubator. The colonies were large, slightly yellow-orange in color, and seemed to cluster together which unfortunately suggested that the plates were contaminated. Thus, the plates were discarded and another attempt at cloning is necessary. In order to proceed, new pNIC-Bsa4 was cut with restrictive enzymes and underwent PCR clean up to yield higher concentrations. Hopefully with a higher pNIC-Bsa4 concentration, a positive clone will result.**
 * Since no colonies grew last time, another attempt at cloning was performed. There were no more reagents to use, so more pNIC-Bsa4 and PCR product needed to be made. The concentrations of the pNIC-Bsa4 cut with restrictive enzymes after PCR clean up were very low. 100 ng of each tube were ran on a gel to ensure that the pNIC-Bsa4 was throughly cut. The gel resulted in positive results because it had two bands in the correct lengths. PCR clean up was performed on eight tubes of PCR squared products. The product of the PCR clean up was then placed in another gel to be used for gel extraction. Performing both PCR clean up and gel extraction caused the concentration of the PCR product to be very low. I proceeded to the cloning process nevertheless.**
 * Good analysis but try to add captions also. Those are important to getting quick information across. Thank you. -Max 10/07/20131
 * __Week of 9/23/13 - 9/27/13__**
 * Katherine - ok keep moving your cloning foward - we want yours to be completed so that you can move forward. - Dr. B 100113**
 * __Week of 9/16/13 - 9/20/13__**
 * __Week of 9/9/13 - 9/13/13__**
 * Primary PCR and Secondary PCR was redone using the oligo mix made in the summer. All the samples were ran on a gel. The primary PCR did not show up on the gel, but the secondary PCR did with the right amount of base pairs. This could have been due to the primary PCR not having enough DNA to show up on the gel, but it had enough to be amplified. Also, Midi-prep was performed on pNIC-Bsa4 pellets. Then it was nanodropped and sent to DNA sequencing for future cloning. The pNIC-Bsa4 resulted in 90% coverage and 98% identity.**
 * . [[image:pnic-bsa4 midi-prepped 9-7-13.jpg width="504" height="311" caption="Figure 1: First nanodrop result of pNIC-Bsa4 after midi-prepped was performed"]][[image:pnic-bsa4 midi-prepped 9-7-13 trial 2.jpg width="504" height="311" caption="Figure 2: Nanodrop results of pNIC-Bsa4 after midi-prep was performed trial 2"]]**
 * The average of the two trials is 83.9 ng/ul. Two samples, one with pLIC-forward primer and one with pLIC-reverse primer, was sent to the DNA sequencing lab.**
 * The blast results are displayed below.**
 * The primary PCR did not show up most likely due to such a low amount of DNA. There is a slightly faint smear near the bottom of lane 2. This may be due to a degrading oligo mix since the mix made during the summer was used. Since it does not take much DNA to be amplified through PCR, the secondary PCR showed results with the right amount of base pairs ~ 900.**

=__**Fall 2103**__=

= = = = = = = = = = = = =__**Week Two: - Katherine -**__= ====__**DNA sequencing result and PCR result? Also - what are your Nanodrops off? (you can put the name of the sample in the Sample ID box so that when you print it - it will already be there). - Dr. B**__====


 * Agarose gel preparation 6/12/13**

=__**Week One:**__=
 * Transformation Efficiency 6/5/13**


 * Nanodrop 6/04/13**