Marianna+U.

__**Week 15 & 16**__


 * Enzyme and Inhibiton assays were performed for YopH enzyme.**






 * Enzyme Assay**






 * Expression, Purification, &** ** Characterization: **


 * As we have move forward with the surrogate protein, YopH enzyme, Option B was chosen, which was started in the early evening at 8 PM. Purification was performed for YopH as well. According to the SDS Page Gel, the results appeared accurate according to the Lane that had Elution 1. This lane had a distinct band, as well as the lane for Elution 2.**




 * Lanes labeled from left to right: **
 * 1: ColorPlus Protein Ladder **
 * 2: Skipped lane **
 * 3 & 4 (same sample): Before IPTG Induction **
 * 5: Soluble Fraction **
 * 6: Flowthrough **
 * 7: Wash **
 * 8: Elution 1 **
 * 9: Elution 2 **









__**Week 13 &14**__
 * The cloning results presented did not show that there was not a positive clone. Therefore,** ** we have move towards the surrogate protein YopH enzyme. I **** did not spend the amount of time in lab needed in order to perform expression of YopH enzyme. Thus, I helped pick up and clean around lab. **


 * __Week 11 & 12__**


 * 11/11/13-11/15/13**


 * After Mini-prepping our eight samples from the week before, each of the samples were Nanodropped to determine which two samples contained the highest concentrations, which would then be sent to the DNA sequencing facility (MBB 1.426). The concentrations for each sample included: **
 * Sample 1- 39.5 ng/uL **
 * Sample 2- 19.5 ng/uL **
 * Sample 3- 12.8 ng/uL **
 * Sample 4- 43.0 ng/uL **
 * Sample 5- 43.4 ng/uL **
 * Sample 6- 16.1 ng/uL **
 * Sample 7-29.0 ng/uL **
 * Sample 8- 12.5 ng/uL **
 * The two samples with the highest concentrations were Samples 4 and 5. Since this was the case, 7 ul of each sample was micropipetted into a 1.7 ul microcentrifuge tube and sent off to the DNA sequencing facility. As we wait for our results, we have started working on virtual screening. We will continue to maintain accurate and precise work, along with ensuring sterile techniques in the wet lab, so we can make progress in our research. **


 * 11/04/13-11/08/13 **
 * This week involved the second and third round of cloning. This consisted of a day to day protocol as we performed both rounds back to back to ensure a positive clone. A master plate was made for both rounds as colonies were chosen to transfer to another plate. Round 2 cloning was Mini-prepped and samples were stored for later Nanodrop measurements. For round 3 of cloning, the pellet samples still need to be Mini-prepped. As soon as it's performed, the DNA sequencing protocol will be utilized to see for a positive clone. **








 * __Week 9 & 10__**


 * 10/28/13-11/1/13**
 * Cohesive end generation was performed as two tubes were assembled. Later, annealing and transformation was performed. Cloning might not have been successful due to the fact that it was left in the incubator at 37 degrees Celsius for over two days. This could cause the cells to be killed. Therefore, a master plate could not have been made. Another cloning attempt will be performed.**


 * 10/21/13-10/25/13**


 * After a successful PCR squared attempt, the sample for PCR squared was taken through the procedure of PCR clean up. The concentration for the PCR squared clean up yielded a concentration of 19.6 ng/uL, which was not as high. There might be some complication later in cloning due to this concentration. After PNIC Bsa4 was prepared and midi-prepped, it was Nanodropped. Yet again, the concentration yielded was approximately 19 ng/uL, which is not enough. The amounts for preparing PNIC Bsa4 had to be amped to higher volumes for ensurance that PNic will be cut correctly. A gel check was performed to show that PNic cut correctly.**







Great captions and analysis. Good job in the wet lab. Do you have any virtual data? Thank you. -Max 10/21/13
 * __Week 7 & 8__**
 * 10/14/13-10/18/13**
 * Secondary PCR and PCR squared were both successful PCR run for the first time.**
 * Secondary PCR, which the samples A (run at 63 degrees Celsius), G (run at 54 degrees Celsius), and H (run at 53 degrees Celsius) were the brightest and all showed promising results of distinct bands. We used a temperature gradient of 10 degrees Celsius, and now we know which temperatures to use if we were to ever re-do Primary PCR. **
 * Although the 100 base pair did not completely and successfully run down the gel, Sample A and Sample H successfully amplified during PCR squared. **
 * We will continue to use sterile techniques in lab, as well as make sure that we are using the correct concentrations, temperatures, and waiting the correct amount of time in order to ensure accuracy and precision in our research. PCR will proceed forward to cleanup. Also, Midi prep was done, but not Nano-dropped yet. Results of the concentration yielded will be put up soon.**


 * __Figure 2:__**
 * 1st PCR squared attempt: Lane 1 was empty, Lane 2 contained 5 ul of the 100 base pair DNA ladder, Lane 3 contained 15 ul of Sample A (run at 63 degrees Celsius) + 5 ul of blue juice (20 ul total), Lane 4 contained 15 ul of Sample G (run at 54 degrees Celsius) + 5 ul of blue juice (20 ul total), Lane 5 contained 5 ul of the 100 base pair DNA ladder, and Lane 6 was empty. **


 * __Figure 1:__**
 * 1st Secondary PCR attempt: The Primary PCR samples used for Secondary PCR were Sample A (63.0 degrees Celsius), Sample G (54.0 degrees Celsius), and Sample H (53.0 degrees Celsius). Lane 1 contains 5 ul of the 100 base pair DNA ladder, Lane 3 contains Sample A from Primary PCR, Lane 4 contains Sample G from Primary PCR, Lane 5 contains Sample H from Primary PCR, and Lane 7 contains 5 ul of the 100 base pair DNA ladder. Lanes 4, 5, and 6 contain 18 ul of one of the Primary PCR samples + 3 ul of blue juice (21 ul total) **


 * 10/07/13-10/11/13**
 * For this week, 16 samples were created to run Primary PCR for the 8th & 9th time. Two different gels were ran with 8 samples in each as each 8 samples were run through a temperature gradient as well as extending the annealing time to 30 seconds in the PCR machine. For the 8th round, the gel was left in for too long, so there was slight contamination where the 8 lanes can barely be seen as well as not being able to see a DNA ladder. They all seem pretty faint bands. For the ninth round, everything seemed just right where a DNA ladder showed as well as a distinct smear in almost every lane of the 8 samples. After many attempts in Primary PCR, we decided to not only try to run our samples on a temperature gradient 10 degrees Celsius, but we also tried to change the amount of dNTP used (2 ul of the 10 mM stock solution, rather than dilute it), and we ran Primary PCR twice, having 16 samples to run on a gel. We used a temperature gradient of 10 degrees Celsius in order to test a +/- 5 degrees Celsius gradient f the original temperature mentioned in the protocol of 58 degrees Celsius. This temperature consisted of temperatures between 53 degrees Celsius and 63 degrees Celsius, including: Sample A: 63.0 degrees Celsius, Sample B: 62.5 degrees Celsius, Sample C: 61.3 degrees Celsius, Sample D: 59.4 degrees Celsius, Sample E: 56.9 degrees Celsius, Sample F: 55.0 degrees Celsius, Sample G: 53.7 degrees Celsius, and Sample H: 53.0 degrees Celsius. **
 * Also, a culture was grown up overnight and then proceeded to MidiPrep in order to yield enough concentration for PNic-BSA4. Unfortunately, there was an error made in MidiPrep where all the PNic -BSA 4 was lost, so another culture had to grown up.**


 * __ Figure 2: __ Primary PCR attempt with a temperature gradient within a 10 degree Celsius range. Lane 1 contains 5 ul of the 100 base pair DNA ladder. Lanes 2-9 contain 18 ul of each Primary PCR sample at a specific temperature + 3 ul of blue juice (20 ul total). The temperatures used were: Lane 2/Sample A: 63.0 degrees Celsius, Lane 3/Sample B: 62.5 degrees Celsius, Lane 4/Sample D: 61.3 degrees Celsius, Lane 5/Sample E: 59.4 degrees Celsius, Lane 6/Sample F: 55.0 degrees Celsius, Lane 7/Sample F: 55.0 degrees Celsius, Lane 8/Sample G: 53.7 degrees Celsius, Lane 9/Sample H: 53.0 degrees Celsius, and Lane 10 was empty. Although the gel did result in amplification in all samples run across the 10 degrees Celsius temperature gradient (53 degrees Celsius- 63 degrees Celsius. Original temperature in protocol: 58 degrees Celsius) **




 * __Figure 1__: Primary PCR attempt with a temperature gradient a 10 degree Celsius range.(53 degrees Celsius-63 degrees Celsius; Original temperature in protocol: 58 degrees Celsius). Lane 1 contains 5 ul of the 100 base pair DNA ladder. Lanes 2-9 contain 18 ul of each Primary PCR sample run at a specific temperature along the 10 degrees Celsius temperature gradient + 3 ul of blue juice (20 ul total). The temperatures used were: Lane 2/Sample A: 63.0 degrees Celsius, Lane 3/Sample B: 62.5 degrees Celsius, Lane 4/Sample D: 61.3 degrees Celsius, Lane 5/Sample E: 59.4 degrees Celsius, Lane 6/Sample F: 55.0 degrees Celsius, Lane 7/Sample F: 55.0 degrees Celsius, Lane 8/Sample G: 53.7 degrees Celsius, and Lane 9/Sample H: 53.0 degrees Celsius. Lane 10 was empty. Although the 100 base pair DNA ladder failed to spread across the entire Lane 1, our samples amplified; showing very bright smears for lanes A, G, and H which will be used for Secondary PCR. **


 * __Week 5 & 6__**


 * 09/30/13-10/04/13**
 * The seventh round consisted of another temperature gradient. in this Primary PCR, nothing seemed to amplify in each of the 8 lanes. More contamination and other things affected this gel. Also it seems that the DNA ladder became contaminated as it ended up appearing in another lane.**




 * 09/23/13-09/27/13**
 * This fourth round of Primary PCR failed. Changes made to the protocol involved using the gradient where the set up involves different temperature changes.**

__Week 3 & 4__
 * 09/16/13-09/20/13**
 * Homology model was made; another Primary PCR was performed by Ashlee Wolf.**


 * 09/09/13-09/13/13 **
 * Another oligo mix was made and tested for a different temperature for the sample. For the sample, the temperature was decreased or increased by approximately 5 degrees. For the first Primary PCR that failed only the times for annealing and elongation were extended to 30 seconds. For the second round of Primary PCR, temperatures were raised about five degrees from the normal protocol along with the extended times. As a result, it also failed.**





__Week 1 & 2__




 * (9/2/13-9/6/13) An oglio-mix was prepared. Primary PCR was preformed and tested on the gel. The results displayed for primary PCR showed that it did not amplify. There can be all sorts of error including: the agarose used to make the gel, the PCR machine used, or contamination in any of the steps taken while making oligo mix or doing PCR. For the next time, changes will be made, such as making another oligo mix and testing a different temperature for the sample. For the sample, the temperature will be decreased or increased by approximately 5 degrees. Safety precaution will be taken into account to ensure a successful PCR. **

__start Fall 2013 above here__

__Week 2:__
 * Marianna - good job. Include your PCR results and your DNA Sequencing result. -- DR .B**

Agarose Gel Preparation(06/13/13)

Post- Midi Prep Kit: Determination of Plasmid(06/12/13)

__Week 1:__

Transformation Efficiency (06/05/2013)


 * Note: Pictures taken after**

Primer Dilutions (06/03/2013)**

Average concentration of pmCHERRY was 119.05 ng/μL Average 260/280 ratio value was 1.91 Average 260/230 ratio value was 2.73 Average absorbance at 230nm was .871

