Nicole+W.+(RP+Summ+14)

**Nicole - nice work. I am eager to see if this enzyme works! - Dr. B 121214**
Include virtual results here too.

12/2/14 ** Lysis: Sonication ** 7:20 pm pre-cool JA-20 rotor - Resuspend in 10 mL of lysis buffer (lysis buffer contents; 50 mM Tris, 300 mM NaCl, 10 mM Imidazole, pH 8.0 with NaOH) - Standard sonication procedure followed for each of 4 tubes (A-D)

**Second Spin Down Step** - Split samples into 2 oak ridges tubes - Centrifuge at 12,900 rpm, 20 degrees Celsius for 30 minutes - Save supernatant

- Syringe filter supertant and pH check to ensure 8.0 **__pH analysis__** Tube A: 8.10 Tube B: 8.17 Tube C: 8.19

- Stored in 4 degree Celsius freezer overnight

12/3/14 ** Purification: ** - 2 mL of flurry added to about 42 mL of supernatant (Resin mixture is a 1:1 ratio of resin to liquid, therefore need to add 1 mL to get .5 mL of resin) - Standard purification protocol followed to execute nickel-column chromatography - Column stripped with water and NaOH, stored with 30% ethanol to cover resin - Sample pun down for 3 minutes at 8,000 rpm in order to concentrate to 1 mL



__**Protein yield of Elution 1:**__ 2.01 mg



__**Purification Conclusion and Analysis:**__ Purification of the protein samples was performed via nickel-column chromatography. In nickel-column chromatography the ability of histidine to bind to nickel was utilized. This histidine is six amino acids at the N terminus of the protein known as the 6X His tag. Nickel is bound to an agarose bead by chelation using nitroloacetic (NTA) beads. First, the flow through step is performed to eliminate waste from the sample and isolate the larger, desired protein within the column where it is bound to and held up by the large nickel bead. The wash step next performed to remove proteins that are only loosely bound to the resin. The final elution step entails using a buffer with a high concentration of imidazole to release the tagged protein from the Ni-NTA resin. The imidazole has a higher affinity for the metal than does histidine, therefore competing with the protein for attachment to the nickel bead. The protein can then be eluted from the column and collected for future experimentation and analysis. Future steps include taking this refined sample immediately to Fast Protein Liquid Chromatography, or FPLC, for further purification. The protein yield of our sample, Elution 1, was 2.01 mg. This value is significantly higher than our previous yield from the first round of purification on this protein. This can be attributed to performing a large scale expression of 2 L instead of the standard 500 mL.

12/4/14 FPLC: - Standard protocol followed to perform FPLC on Pv6PG sample





__** FPLC trial #2 Conclusion and Analysis: **__

Fast Liquid Protein Chromatography, or FPLC, may be executed after purification and sonication as a method of separating the mixture into its constituents and further purifying the protein. FPLC specifically refers to size-exclusion chromatography. The sample is first concentrated in a spin tube concentrator to about 1 mL. This 1 mL volume is optimal because the FPLC machine tube can hold 2 mL maximum and using an amount about half of that will ensure not as much is lost in the process. This instrument has a molecular weight cut off of 10 kDa meaning that any molecule weighing that much or above is conserved in the top portion of the concentrator above the membrane. The weight of Pv6PG measured to be 53 kDa, well above this 10 kDa spin tube concentrator cut off. In addition, FPLC buffer is made and degassed to remove any possible air bubbles. To perform purification through FPLC the loop is first flushed with 10 mL of freshly made buffer to clear the column of any extraneous contaminating proteins. Next, the sample is run through the G75 column. Lastly, the resulting graph is analyzed on the computer and utilized to determine where the desired protein is located after elution through the column. The FPLC column itself separates components of a sample through both a stationary and mobile strategies. The stationary component is composed of spherical agarose gel beads that are inside the column. These beads contain pores that trap smaller proteins as they travel down the column and slow them down. The mobile component refers to the flowing buffer liquid that elutes larger proteins through the column faster. As the larger proteins are excluded from the pores in the agarose gel beads the general trend in the end is that proteins leave the column in a pattern of largest first to smallest last. The computer then measures concentrations and illustrates these values on a graph with peaks corresponding to a higher concentration, or presence of protein in the solution. In our trial of FPLC we received a peak on the graph at approximately 50-55 mL volume on the X axis. This peak when measured against the standard indicated a protein size of Pv6PG, which is about 53 kDa. The size of this peak however is small overall, ranking at about 20 on the Y axis. It is possible that additional rounds of large scale expression and purification may need to be performed to secure a larger amount of protein that can be utilized for future trials of enzyme and inhibition assays. Until then tubes 31-34 were isolated from the carousal of the FPLC machine, reconcentrated to 1 mL and stored until next semester. The chosen method of storing this sample was snap freezing to ensure effective long term storage.

11/22/14 No wet lab procedures performed this week - Thanksgiving Week

11/17/14 **Making LB media** (for next rounds of protein expression) - need 2 x 2L for 2 simultaneous large scale protein expressions - Follow standard LB protocol; multiply dry ingredients by 4 for EACH 2L flask

__**For 2L LB**__ - 20g Bacto-tryptone - 10g Bacto-yeast extract - 20g NaCl

- Combine in 4L flask with 2000 mL of dH2O - Stir until dissolve completely with stir bar - Autoclaved

11/18/14 Transformation with BL21 cells - Follow transformation protocol with BL21 cells - Combine 50 ul cells with 1 ul plasmid



11/19/14 **Making 3 x 50 mL and 3 x 100 mL overnight cultures** - Each flask contains appropriate amount of LB, kan, and colony sample on pipette tip

11/10/14

Week 11
11/4/14 - In DIY diagnostics lab large centrifuge
 * Spin Down Step**

- Stored at 4 degrees Celsius 1-2 days
 * Syringe Filter Step**

11/6/14 **Analysis and Conclusion of Protein Expression:**A culture of the successful was clone grown overnight in a flask of LB to receive a high level of bacterial growth. Midi-prep of this transformation was performed to extract the DNA from a spun down sample of this culture to be utilized for future experiments. To initiate protein expression an overnight transformation was performed in the lab in order to incorporate the successful clone into BL21 (DE3) cells. These cells facilitate protein expression of the genes that are encoded upon the combined plasmid. This mixture was plated on agar-solidified plates containing minimal media and the resulting colonies were utilized to perform Option A protein expression. Option A required growing small 4 mL cultures of LB media in transformation tubes combined with 4 ul of kanamycin antibiotic and a sample of the clone colony on a pipette tip. These cultures were placed in the shaking incubator for approximately 8 hours and inspected for a cloudy resolution that indicated successful bacterial growth. These cultures were introduced into a flask of 500 mL of LB media until a final point of 0.043 absorbance was measured by the spectrometer. The desired absorbance point was 0.1 but regardless the flask was placed in the shaking incubator and absorbance was measured periodically until a final reading of 0.512 was reached two hours later. At this point IPTG was added to the mixture to induce protein expression. The mixture was allowed to shake at room temperature for 20 hours at which point it was spun down. The supernatant was discarded because it consisted of extraneous components such as LB media while the protein-containing pellet was kept. The pellet was lysed or broken open, and the process of sonication was performed to further release the proteins from the host cells. Again the solution was spun down but this time the supernatant was kept and the cell-debris containing pellet was thrown out. These processes were performed to utilize the bacterial cells to express the manipulated vector and produce a vast amount of protein that will be utilized later on in wet lab processes. Protein expression occurs after gene expression and consists of the stages after DNA has been transcribed into messenger RNA. The mRNA is then translated into polypeptide chains which are ultimately folded into proteins. **Protein Purification**- Samples 0 & 1 go in Julio- Samples 2-6 go in 4 degree Celsius fridge- We used a new nickel column - saved and stored in 4 degree Celsius fridge for future use



**Protein Yield:**__0.81 mg/mL (average__)

11/7/14 FPLC- Fast Liquid Protein Chromatography ***Gel filtration chromatography (size exclusion chromatography) - Maximum pressure of column at 0.2 MPa - ours was at 0.14 MPa - Our protein: ~53 kDa



Week 10
10/28/14 7:38 pm - get out BL21 cells from -80 freezer 7:42 pm - Combine 50 ul BL21 with 1 ul plasmid 7:45 pm - Let incubate on ice for 30 minutes 8:15 pm - Heat shock 42 degrees Celsius for 45 seconds 8:17 pm - Incubate on ice for 10 minutes 8:27 pm - Add 250 ul pre-warmed SOC - Incubate in 37 degree Celsius shaking incubator for 1hour 9:27 pm - Plate 200 ul on one LB agar+kan+sucrose plate and 50 ul on another 9:28 pm - Place plates in 37 degree Celsius incubator overnight
 * Transformation into BL21 (DE3) cells:**

10/29/14 - check plates after overnight incubation in 37 degree Celsius incubator - at 9:00 am plate A had a large amount of small colonies but plate B had almost no growth - Let incubate until 2:30 pm; both plates had numerous small colony growth, plate A had many more and some were clustered together while plate B had more distinct, separate colonies



10/30/14 10:45 am - Begin growing small cultures; 4 mL of LB in transformation tubes with 4 ul of kan and a pipette tip containing sample colony from plate B - I made 12 small transformation tubes - 48 mL of solution total 11:29 am - Transformation tubes with finished mix placed in 37 degree Celsius shaking incubator for 8 hours
 * Option A Protein Expression:**



7:00 pm
 * Pre-warm 500 mL LB

7:30 pm - Take tubes out of incubator - Initially add 8 mL (2 tubes worth) of transformation solution to 500 mL flask of LB, measure with spec to receive absorbency measurement: 0.00 - Additional 12 mL added: 0.00 - Additional 12 mL added: 0.006 - Additional 16 mL added (remaining amount of transformation solution): 0.043 - Placed in incubator at 8:20 pm, checked periodically until at about 0.5

10:20 pm - Absorbency reading: 0.512 - 271 ul of IPTG added to turn on protein expression - placed in room temperature shaking incubator at 10:48 pm for 18-20 hours

10/20/14 10:45 am - separate into 4 50 ml conical tubes and get weight within 1 gram of each other - centrifuge for 15 minutes; 6,000 x g, 15 minutes, 4 degrees Celsius - discard supernatant
 * Midi Prep:**

11:29 am Followed Sigma-Aldrich Midi-prep procedure to elute DNA - Nandrop to receive concentration and store in -20 degrees Celsius refrigerator



10/19/14 - LB pre-warmed in 37 degree shaking incubator (for at least 30 minutes) -160 mL LB in 1000 mL flask (no 500 mL flasks available) - 160 ul kan - sterile pipette tip used to extract colony sample and released into the LB/kan mixture - colony used was Colony F - 2:8 ratio of vector:insert (positive clone), taken from master plate - LB + kan + colony allowed to shake overnight in the 37 degree shaking incubator to allow for wide-scale bacterial growth
 * Overnight transformation: (for midi-prep) **

10/15/14 - in preparation for overnight transformations to proceed with protein expression - autoclaved, sealed and stored in fridge
 * LB media made:**


 * Next Step:** Move on with successful clone to protein expression, purification and characterization, will start on October 15, 2014.

10/6/14

__**DNA sequencing results:**__ (received on October 4, 2014, over the weekend) Successful clone received for sample NBWFF/NBWFR - plate with 2:8 ratio of vector:insert



Rest of week no new wet lab work was performed - waiting for DNA sequencing results to come back.

10/1/14

Samples delivered to DNA sequencing facility for sequence verification. - change: I used 500 ng of DNA template instead of 300 ng

9/30/14

DNA samples prepared to be sent in for DNA sequencing - stored overnight and will be submitted tomorrow (10/1/14).

9/29/14


 * __Qiagen mini-prep of colony samples A-I:__**

10:28 am - 200 ul resuspension solution - 200 ul of lysis solution
 * Step 1:** Harvest and lyse bacteria

10:35 am - 350 ul of neutralization solution - 12 x g used for each centrifugation
 * Step 2:** Prepare cleared lysate

- 500 ul colum preparation solution to binding column of a collection tube - centrifugation at 12 x g
 * Step 3:** Prepare binding column

10:43 am - transfer cleared lysate into binding column - centrifugation and discard flow-through
 * Step 4:** Bind plasmid DNA to column

10:51 am - 750 ul wash solution - 2 x centrifugation at 12 x g
 * Step 5:** Wash to remove contaminants

11:07 am - transfer to new collection tube - 30 ul elution solution
 * Step 6:** Elute purified plasmid DNA

Nanodrop of Samples A-I: A: 77 ng/ul B: 49 ng/ul C: 54.55 ng/ul D: 58.45 ng/ul E: 87.40 ng/ul F: 79.70 ng/ul G: 70.15 ng/ul H: 96.95 ng/ul I: 54.55 ng/ul
 * Average concentrations found to be (after 2 trials);**

Week 5
9/26/14


 * __pNIC-Bsa4 LIC Cloning Analysis: __**

pNIC-Bsa4 LIC cloning enables the insertion of a PCR amplified gene into a plasmid vector for DNA replication or protein expression. LIC cloning stands for Ligation Independent cloning, indicating that no DNA ligase will be required to catalyze covalent bonds in this cloning procedure. In this case the gene of interest would be considered the insert and the pNIC-Bsa4 would be considered the vector. The overall goals of this process are to successfully insert the gene of interest into the pNIC-Bsa4 vector, generating cohesive ends with T4 polymerase and completing annealing and transformation. Both the insert and the vector require preparation before cohesive end generation can be performed. For the insert this includes primary PCR, secondary PCR, PCR squared and PCR clean up. The vector preparation includes the excision of the sacB gene. This step is crucial because in the presence of sucrose sacB codes encodes for an enzyme called levansucrose which will convert sucrose into a toxic byproduct that inhibits any cell growth. SacB is removed with the restriction enzyme BSAI in a water bath procedure and verified on an agarose gel. If the BSAI cut correctly after electrophoresis the agarose gel will show two separate DNA bands, one slightly below the 2,000 bp mark and a larger one around the 6,000 bp mark. Next, in cohesive end generation T4 polymerase is utilized to create complementary base pair overhangs on both the insert and the vector. On the vector dGTPs are used so that the T4 will stop excising, or get ‘stuck’, at the first C base that it encounters. On the insert the same process occur but dCTPs are used in the mixture so that the T4 will get stuck at the first G base. The overhangs on the vector and insert consist of about 20 bases each and will hydrogen bond to each other due to the complementary nature of the base pairs. Because the overhangs are so long the hydrogen bonds will be enough to hold the two pieces together without requiring any DNA ligase to catalyze the covalent phosphodiester bonds between the gene of interest and the pNIC-Bsa4. Next, in the annealing step different ratios of the prepared PCR insert and the prepared vector are mixed together in round bottom transformation tubes. This step “closes” the plasmid by integrating the target gene into the pNIC-Bsa4 where the SacB gene used to be. The general time allowance for this step is about 16 hours although it may take longer than that for colony growth to appear. During transformation DH5 alpha competent cells are added and the solution is heat shocked to create pores in the DH5 alpha cells and allow them to incorporate the foreign DNA into their own. Following this the tubes are placed back on ice and incubated at 37°C for 1 hour to kickstart growth. The varying ratios are then plated on LB agar + kan + suc plates overnight and inspected for colony growth the next day. The sucrose in this step is required to ensure the removal of sacB. A master plate is made the next day with about 9 colonies from the different plates to serve as an archive in case one of the colonies is confirmed to be a successful clone. Small overnight cultures were made consisting of 5 ml LB + 5ul kan with the pipette tip containing colony sample. Cloudy resolution of liquid indicates successful colony growth. The sample were spun down and mini-prepped in order to extract the DNA from each sample colony and finally nanodropped and sent to DNA sequencing.

9/25/14

__** Next round of pNIC-Bsa4 cloning start: **__ I made a number of __changes__ to the protocol after research online - to compensate for having a large gene insert.

- incubate at room temperature after addition of T4 polymerase for __45 minutes__ - heat inactivate for __30 minutes__
 * Cohesive End Generation **

- incubate __20 minutes__ at room temperature after combining ratios - directly following previous step, leave on ice for __10 minutes__ - after adding DH5 alpha cells incubate on ice for __1 hour__ - heat shock for __90 seconds__ at __37 degrees Celsius__
 * Annealing and Transformation **

9/22/14

__**Qiagen PCR clean up of PCR squared samples:**__

Week 4
9/15/14

__**Previous Cloning Round Analysis/Conclusion/Future Steps:**__I received successful colony growth in each of the squared plate sections. If cloning had proceeded successfully I would have used this plate as an archive to obtain future samples of my clone. Overall however this round of cloning failed after reviewing my DNA sequencing results. The highest percentage I received was 14% identity of all my samples. After a comparison of my desired sequence and the FASTA sequence I received from DNA sequencing I was able to see that the only ‘copied’ part was the very beginning and end of my sequence; therefore the desired gene was not inserted into the vector at all and my sequencing results seems to only consist of the pNIC vector. My next step is to go back to my secondary and make more PCR squared samples. I plan to do one round with gel extraction and a simultaneous round under the normal protocol procedures without gel extraction – only PCR clean up. I will then proceed to more rounds of cloning with these purified samples.

I performed 3 simultaneous PCR squared procedures. Two of these will be used to perform a gel extraction (requires double the normal amount of PCR squared sample) while the remaining batch will be utilized in the normal procedure - will be run on a gel, PCR cleaned and prepared for vector insertion if the gel results are successful. I hope to proceed to another round of cloning with both the normal and gel extracted PCR samples to see if this is the factor effecting the inhibition of my gene into the pNIC vector.
 * __Today:__**

Expiration dates of reagents: Q5 Buffer - 3/17 dNTP - 3/16 Q5 Polymerase - 5/15

- PCR squared run on Lamborghini for all samples - at 55 degrees Celsius

__**DNA sequencing results** **received:**__ __**- insert pictures here**__
 * 9/11/14**


 * 9/9/14**

[[image:vdsstream/nw5248_NikkiW_091514_masterplate_results_pic.JPG]]
__**Cohesive End Generation of PCR Inserts and Accepting Vector:**__ __**- Note:**__ use NEBuffer 2 for cohesive end generation T4 polymerase buffer
 * 9/8/14**
 * 9/7/14**
 * Preheat SOC media and plates (LB agar + kan + suc) ahead of time, also turn on water baths to 42 degrees Celsius and 75 degrees Celsius

__Labor Day__
 * 9/1/14**

PCR squared performed with my secondary PCR sample from 8/29/14. I utilized these PCR squared samples to perform a PCR clean up to prepare for generating cohesive ends on the inserts. Subsequent nanodrop after clean up.
 * 9/2/14**


 * __INSERT PCR SQUARED AGAROSE GEL RESULT PICTURE HERE__**



__**Preparation of pNIC-Bsa4 as Accepting Vector:**__ - See 7/18/14 for same procedure followed - Double amounts indicated in protocol to obtain a total of 50 ul total in each tube (to compensate for large amount of pNIC plasmid required) - 2.25 ug of pNIC-Bsa4 plasmid = 2250 ng - This number divided by the concentration of the plasmid will give the appropriate number of ul of pNIC plasmid to add > >2250 ng (1 ul / 107.9 ng) = 20.85 ul - Water needed: 22.65 ul __**- Note:**__ use NEBuffer 4
 * 9/3/14**

__**INSERT AGAROSE GEL PICTURE HERE**__




 * I accidentally performed PCR clean on my PCR squared sample for a second time, meaning to do the process on my cut pNIC samples instead. I finished the process however once I realized my mistake because it was too late to go back and nanodropped the samples again to ensure sufficient concentrations.

__**Gel Extraction of gel bandsSample #3**__ - Performed same process as on Gel Extraction Samples #1 and #2 - Combined Gel extractions #3A and #3B into one large sample __- Change*__**: Eluted with 25 ul of preheated elution solution instead of 50 ul in an attempt to get a higher concentration**
 * 8/29/14**



- Above is a sample graph of what the typical GE #3 sample looked like. Altogether I had 6 separate samples for this gel extraction but all graphs look similar to this one.


 * Results (GE#3):** This round of gel extraction is concluded to be a failure and produced even worse results than the first two separate extractions I performed; the concentrations were very low and after analyzing the nanodrop graphs I can see that there was seemingly no DNA present in my samples at all. Otherwise, I would have received a shift in the nanodrop graph with a maximum yield placing at the 260 nm wavelength. It is possible that I missed a step in the procedures or not changed the filter properly in between the wash and elution steps.

After receiving failing results with my multiple rounds of gel extraction I decided to go back to the beginning and begin a new round of primary PCR, utilizing my already prepared oligo mix of Pv6PG.


 * Rerun of Primary/Secondary PCR Pv6PG:**


 * __INSERT AGAROSE GEL RESULT PICTURE HERE__**

I received a "smear" for my primary PCR and a strong band for my secondary PCR placed right below the 1.5 kb bp mark. These are indicative of successful results, I will take my secondary PCR sample and move on to PCR squared tomorrow.

Gel Extraction of gel bands Sample #2
 * 8/28/14**
 * - Performed same process as on Gel Extraction Sample #1 on 8/27/14**

Gel Extraction: * Turn on water bath to ~55 degrees Celsius ahead of time / preheat Elution Solution to 65 degrees Celsius using water bath 1. Excise band: be very sterile! - Clean UV light platform with ethanol and lay seran wrap over it - Use brand new razor; make cut clean cut then turn off the light and clean the blade with ethanol in between each new cut > >attempt to minimize exposure of gels to the UV light > >wear appropriate protective equipment to protect against UV rays; gloves, glasses, lab coat, and head/face gear 2. Weigh gel
 * 8/27/14**

__**Gel Extraction # Weight (g) Weight (mg)**__ 1 .45 g 450 mg 2 .29 g 290 mg 3A .44 g 440 mg 3B .43 g 430 mg

3. Calculate gel solubilization solution amounts (ul):
 * 100 mg agarose gel / 300 ul gel sol. = weight (mg) / X ul gel sol.**

1 1350 ul 2 870 ul 3A 1320 ul 3B 1290 ul
 * __Gel Extraction # Gel Sol. Amount (ul)__**

4. Prepare column with 500 ul column binding solution each 5. Centrifuge 1 min 12,000 x g 6. Add gel sol. to extracted gel band samples - incubate 10 minutes in 50-60 degree Celsius water bath (mine ~55 degrees Celsius) 7. Add isopropanol (one gel volume - the weight of original band, 1 ul:1 mg) 8. Add 700 ul wash (Centrifuge x 2) 9. Elution with 50 ul of preheated Elution Solution - Above is a sample graph of what the typical GE #1/2 sample looked like. All graphs look similar to this one, yielding very low concentrations overall.
 * Perform this same process for Gel Extractions #2, and #3A-B


 * Results (GE #1 & #2):** For these samples I received very low concentrations with the highest being about ~20 ng/ul. While a few of the graphs did exhibit a peak at 260 ng/ul, I do not believe these samples are strong enough to be taken into the cloning rounds. If I did choose out of these samples I would take the highest concentration and possibly consider running it through the PCR machine again to encourage further amplification of my desired DNA.

Nanodrop of old pNIC sample from last midi prep.
 * 7/18/14**





Cut pNIC-Bsa4 with with Bsa in order to remove SAC-B gene - 2.25 ug of pNIC-Bsa4 plasmid = 2250 ng - This number divided by the concentration of the plasmid will give the appropriate number of ul of pNIC plasmid to add > >2250 ng (1 ul / 81.9 ng) = 27.4725 ul > >2250 ng (1 ul / 76.5 ng) = 33.33 ul - Double the original amounts on the protocol to compensate for this ul amount of pNIC; should have 50 ul total in one tube of pNIC to be cut - Place sample in water heated PRE-HEATED to 37 degrees Celsius for 3 hours - Perform PCR clean up, elute with 30 ul of elution solution, NOT 50 ul (pNIC needs to be a higher concentration) - check on agarose gel
 * Started to prepare pNIC-Bsa4 to be an accepting vector:**
 * I used two different pNIC plasmids to cut

__**INSERT AGAROSE GEL PICTURE HERE**__

- Nanodrop to obtain final concentration after PCR clean up - Save in Julio (-20 degrees fridge)









Secondary PCR with primary PCR mix from 7/16
 * 7/17/14 **

PCR^2



PCR clean up

Prepare overnight culture of pNIC-Bsa4: - 160 mL of LB broth + 160 ul kan (1:1 ratio) > LB must be warmed in incubator for 30 minutes prior to use - Swipe pipette tip over pNIC colony and insert into flask of WARM LB - Incubate 16 hours overnight > started incubation about 6:30 pm, will come back around 10 am-12 pm







Making Oligo mix: Redo
 * 7/16/14**

Primary PCR: redo

** 7/15/14 **
2 more runs of PCR with temperature gradient (at night) - Same temperature range (for both blocks) - Time of annealing step: 20 seconds (for both blocks) - Time of elongation step: 40 seconds on block A and 50 seconds on B > Did not have enough template to put two ul in each; only had enough of my primary left to make 8 sample on block A and 5 on block B > > Saw some bands form on both of these gradients > > Bands were not intense enough though; will redo my olgio/primary, and use that to make another secondary PCR under these condition (longer elongation time). Theoretically the Primary PCR could have been degraded because it has been taken out/used so many times.
 * Results: successful to an extent!







2 runs of PCR with temperature gradient: - Temperature range: 55-63 degrees Celsius - Time of annealing step: 20 seconds on block A and 30 seconds on block
 * Results: unsuccessful






 * correction in caption above: should read runs 24-31

Next step: - Increase time of ELONGATION STEP (after annealing step) - Keep annealing time increased also - Keep temperature gradient from today - Double DNA concentration; add 2 ul of template instead of 1 ul

** 7/14/14 **
Secondary PCR: Temperature gradient again with 5 samples (PCR 11-15) - SAME temperature range as before, still with 5 samples but increased time of the annealing step to 20 seconds *Results: unsuccessful

Secondary PCR: Temperature gradient, with 5 samples (PCR 6-10) - Tube A: 58.5 degrees Celsius - Tube B: 59.1 degrees Celsius - Tube C: 60 degrees Celsius - Tube D: 61.1 degrees Celsius - Tube E: 62.6 degrees Celsius
 * Results: unsuccessful





4/5 Runs of Secondary PCR: We are not trying different temperatures and times for the annealing step to see if this is the reason our secondary is not working. We have a very large target (1400 BP) so it may need a longer amount of time to amplify the DNA.

- in first tube (A) will alter TEMPERATURE from 58 degrees to 65 degrees - in second tube (B) will alter TIME from 10 seconds to 30 seconds
 * Results: Both of these were unsuccessful.



** 7/11/14 **
Visit to Pickle Research Center.

Third run of Secondary PCR and agarose gel:

- We have decided to change certain conditions when running next secondary PCR to try and find what is causing it to fail, will isolate one variable at a time > change temperature from 58 degrees to 65 degrees > increase time during amplification step - Will come in Monday morning (7/14/14) and run these two PCR's simultaneously

** 7/10/14 **
Primer Dilution: Order Number; 10501455 Reference Number: 123210405 (forward) and 123210404 (reverse) Name; FtAcpa-for/rev, ***when I ordered these I accidently named them wrong, names should read Pv6PG forward/reverse

- For stock solution use spreadsheet t calculate amount of TE to add (found on Google Docs in Misc/Primers folder) > my amount = 281 ul of TE combined with powder at bottom of tube received from IDT - For working dilution combine 40 ul of the stock solution with 160 ul of Tris in 1.7 mL tube to give a total volume of 200 ul > Tris maintains the pH and allows for the primer to last longer > Be sure to label working dilution "20 uM working dil for/rev NBW date"


 * We ordered extra primers, was a mix up because of name confusion. Will store and use if need extra.

(INSERT PICTURES OF BOTH SPEC SHEETS)

First run of Secondary PCR: unsuccessful results



Second run of Secondary PCR: unsuccessful results



** 7/8/14 **
Notebook catch up day.

** 7/7/14 **
Restriction enzyme digest: - NEBuffer 2.1 works for both - Follow given protocol posted on google docs - Original concentration of plasmid = 304.2 ng/ul, to get 1500 ng of DNA need 4.93 ul of plasmid - Lane 3 note*** combine 3 ul of plasmid, 2 ul of blue juice and 7 ul of water



First run of Primary PCR and agarose gel:



(ADD UV LIGHT PICTURE OF PRIMARY PCR GEL 1)

Protein Characterization with Zain continued: (FINISH INSERTING NOTES AND PICTURES HERE)

** 7/3/14 **
Second pNIC PCR and agarose gel run: (sucessful!) - pNIC initial concentration = same as before > > Use same dilutions as before - Primers used; same pLIC primers (2.5 uM concentration) - Each sample tube had 26 ul total (***change amount of DNA template in each tube to double, or 2 ul) > > TUBE A: 23 ul master mix, 2 ul DNA (Dilution 3), 1 ul TAQ > > TUBE B: 23 ul master mix, 2 ul DNA (Dilution 2), 1 ul TAQ > > TUBE C: 23 ul master mix, 2 ul DNA (Dilution 1), 1 ul TAQ > > TUBE D: 23 ul master mix, 0 ul DNA add 2 extra ul of DDW), 1 ul TAQ - Used 1 kb DNA ladder (5 ul in first lane) - Add 5 ul blue juice to each sample and pipette up and down to mix - Inserted 20 ul of each sample into subsequent lanes

(INSERT PNIC PCR #2 PICTURE HERE)

Nickel Column purification: - Flow through step - Wash step - Elution step - Started characterization process > made home-made page gel and stored overnight



- After nickel column purification we performed a nanodrop on the Elutions 1&2 samples to determine the corresponding concentrations.











First pNIC PCR and agarose gel run: (unsucessful) - pNIC initial concentration = 46.25 ng/ul > Dilution 1 (Tube C): 1:10, 1 ul of stock and 9 ul of DDW > Dilution 2 (Tube B): 1:100, 1 ul of Dilution 1 and 9 ul of DDW > Dilution 3 (Tube A): 1:1000 ul of Dilution 2 and 9 ul of DDW - Primers used; pLIC forward/reverse in 2.5 uM stock concentration - Each sample tube had 25 ul total > TUBE A: 23 ul master mix, 1 ul DNA (Dilution 3), 1 ul TAQ > TUBE B: 23 ul master mix, 1 ul DNA (Dilution 2), 1 ul TAQ > TUBE C: 23 ul master mix, 1 ul DNA (Dilution 1), 1 ul TAQ > TUBE D: 23 ul master mix, 0 ul DNA add 1 extra ul of DDW), 1 ul TAQ - Used both 100 bp and 1 kb DNA ladders (5 ul) - Add 5 ul of blue juice to total sample and pipette up and down to mix - Pipette 20 ul of sample into each lane
 * 7/2/14**

(INSERT PNIC PICTURE HERE)

Continuation of practice run of Fthap protein expression: - Sonication to further break open cells (in Biotech lab) - Spin down in large DIY centrifuge (cool down ahead of time before sonication) > 13,500 rpm, 4 degrees Celsius - Checked pH of samples and added nickel in preparation for purification the following day.

DNA Sequencing Facility Results: Midi Prep
 * 7/1/14 **









Start protein expression with Zain : utilized overnight culture; > > protein; FtHap > > plasmid; pNIC-Bsa4 > > grown in; BL21 (DE3) cells - overnight culture grown on 6/30/14 and spec procedures continued on 7/1/14 - Computer:Asparate - Spectrometer:Chipper (used Chipper and LoggerPro software in order to determine the OD600 values) - Combine 500 mL of warm LB media (put in incubator ahead of time), 500 ul of Kanamycin resistance (kan = 1:1 ratio), and 10 mL of overnight culture mix (use sterile, packaged pipettor with bulbs!) > > measure OD600 value > > desired OD600 value is 0.1 - Keep adding increments of overnight culture mix and check concentration via spectrometer > > total added at 12:49 pm: 25 mL to reach an OD600 of 0.90 - Begin incubating for 30-45 minute periods and check OD600 periodically > > desired OD600 is 0.5 - My time increments: > > 2:19 pm: 0.115 > > 2:47 pm: 0.135 > > 3:46 pm: 0.683, added 259.5 ul IPTG at this point to turn on protein expression then incubate for 4 hours; *note, sample was over concentrated with bacteria (more than 0.5 desired value) - Spin down in centrifuge - Discard LB liquid and add lysis buffer to pellet and resuspend - Store in -80 fridge overnight



FPLC Observation of other group's samples: (INSERT FPLC GRAPH HERE)

Making Oligo Mix: - Follow instruction in Figure 7 to find out necessary info to make mix



- Plate: B - Wells: Pv6PG_1-Pv6PG_36 (36 total) - Position: D7-G6

- Be very sterile with this procedure - First use P200 to mix each well individually up and down (use different tip EACH time) - Take P2 and transfer 1 ul of each well into a 1.7 mL tube - Total should be 1 mL so subtract 100 - your number of well to get the number of ul of water > Ex; I had 36 wells so I added 64 ul of water to get a total of 100 ul or 1 mL (100 - 36 = 64)

6/30/14
Primer Design Tails: - Purpose of this computer lab exercise was to design a forward and a revers primer for PCR amplifying the CDS of your gene interest sequence and synthesizing compatible ends suitable for Ligation Independent Cloning (LIC) for insertion into the pNIC-Bsa4 as the acceptable vector for eventual expression





6/27/14
Visit to TACC facility: TACC stands for the Texas Advanced Computing Center. This facility designs/deploys the most powerful technologies in the world to help researchers make discoveries that change the world.







- Figures 1-3 show Stallion: a 328 megapixel tiled-display > large 16x5 tiled display of 30 inch Dell monitors > this configuration allows for exploration of visualizations at an extremely high level of detail and quality > the cluster allows users access to over 82 GB of graphics memory and 240 processing cores



- Figure 4 shows Lasso: multi-touch display > 12.4 megapixels resolution > 6 46" monitors in 3x2 grid configuration > driven by a single node



- Figure 5 shows Mustang and Silver: Stereoscopic 3D visualization systems

Submitting DNA ti DNA sequencing Facility: Midi Prep - Average concentration: 73.75 ng/ul - To find amount of DNA to add to mix; > 73.75 ng/1 ul = 300 ng/X ul (isolate X), X = 4.07 ul - Primer; dilute to 10 uM by mixing 1 ul stock primer with 9 ul Tris (Tris helps mantain pH better than water) - To make 12 ul mix total; 4.07 ul DNA + 1 ul diluted primer + 6.93 ul nanopure H2O - Submitted to http://coreweb.icmb.utecas.edu/ > order number; 104811 > names; NBW1 (Sp6) and NBW2 (T7)

Continuation of practice run of Fthap protein expression: - Sonication to further break open cells (in Biotech lab) - Spin down in large DIY centrifuge (cool down ahead of time before sonication) > 13,500 rpm, 4 degrees Celsius

6/26/14
Practice run of protein expression: utilized overnight culture; > > protein; FtHap > > plasmid; pNIC-Bsa4 > > grown in; BL21 (DE3) cells - Computer:Asparate - Spectrometer:Chipper (used Chipper and LoggerPro software in order to determine the OD600 values) - Combine 500 mL of warm LB media (put in incubator ahead of time), 500 ul of Kanamycin resistance (kan = 1:1 ratio), and 10 mL of overnight culture mix (use sterile, packaged pipettor with bulbs!) > > measure OD600 value > > desired OD600 value is 0.1 - Keep adding increments of overnight culture mix and check concentration via spectrometer > > total added at 1:19 pm: 30mL to reach an OD600 of 0.93 - Begin incubating for 30-45 minute periods and check OD600 periodically > > desired OD600 is 0.5 - My time increments: > > 2:19 pm: 0.115 > > 2:50 pm: 0.135 > > 3:31 pm: 0.258 > > 3:47 pm: 0.43 > > 4:02 pm: 0.404, added 260.5 ul IPTG at this point to turn on protein expression then incubate for 4 hours - Spin down in centrifuge - Discard LB liquid and add lysis buffer to pellet and resuspend - Store in -80 fridge overnight

6/25/14
Ran agarose gel of 3rd PCR; results were successful!
 * 3rd PCR Results on pGBR22:**
 * Note: changed amount of agarose powder when making gel from 0.7 grams to 0.8 grams per 70 mL of 1x TAE buffer.



**6/24/14** I ran my third PCR on pGBR22 today. Utilized previously made samples of diluted primers (M13 forward and reverse) and template (1:100 and 1:1000 PGBR-22). Gel will be run using these samples tomorrow (Wednesday 6/25/14).
 * PCR (#3)**

Goal was to determine the DNA sequence of a plasmid.
 * 6/23/14 **
 * Analyzing DNA sequence **



Found reverse complement and compared to M13 Rev. First found TA and marked the flanking regions on either side. Inserted fragment of M13 Rev into the reverse compliment in between the two flanking regions (underlined). Also highlighted the start codon, stop codon and Shine-Dalgarno sequence.

** Agarose gel **
To make 1% agarose gel; - combine 70 mL 1 X TAE buffer and 0.7 g agarose. *DO NOT use 50 x TAE buffer, dilute to 1 x if necessary. - microwave for about 40 seconds (should boil but not over top) then cool under faucet water (hold flask with a protective mit) - add ethidium bromide (cancerous,* only use in confined area and use pipettors in back of lab, dispose of gloves immediately after handling) - then pour into cast and allow to harden

Prepare samples; - add appropriate amount of blue juice to each sample (we added 5 ul to each of our 25 ul samples) (be sure to differentiate between protein blue juice and DNA blue juice)

- once gel has hardened prepare all equipment, load ladder in first lane then each sample in consecutive lanes (we added 20 ul of each sample per lane) - remember DNA flows from negative to positive (red = positive)

I ran my PCR samples (from 2nd PCR because first failed) The resulting gel was not successful because no individual bands were observable for any of the lanes. Will do PCR for a third time and rerun gel.

** Midi prep **
Following midi prep next step is to DNA sequence and nanodrop to determine concentration.



FtHap in 2 x 125 mL flasks. Performed LB culture overnight using plates grown last week. Combined LB/antibiotic in flask, used pipette to pick up single colony and dispense into flask. Allowed overnight for bacterial growth to happen. (Cloudiness of solution next day is indicative of significant bacterial growth)
 * 6/18/14**
 * Protein Expression**

harvest by centrifugation; spun in large centrifuge in DIY lab. Trash the liquid (left over LB) and keep the pellet at this point (where the protein is). Lyse to break open the cells and then spin down again. Now we want to the supernatant (soluble parts) and to discard the pellet (cell debris).

PCR of PGBR-22 using M13 forward/reverse primers.
 * 6/17/14: My First PCR**

Dilute primers twice: 2 ul stock and 8 ul water THEN 7.5 ul diluted and 52.5 ul water Dilute template: 1:100 and 1:1000

To make Master Mix: combine 15 ul Thermopol buffer (10x), 3 ul dNTP, and 6 ul of each f the diluted forward and reverse primers.


 * Tube A:** 5 ul MM, 1 ul 1:1000 template, 18 ul water, 1 ul diluted Taq
 * Tube B:** 5 ul MM, 10 ul 1:1000 template, 9 ul water, 1 ul diluted Taq
 * Tube C:** 5 ul MM, 10 ul 1:100 template, 9 ul water, 1 ul diluted Taq
 * Tube D:** 5 ul MM, 0 ul template, 19 ul water, 1 ul diluted Taq
 * should have total of 25 ul in each tube!

Week 1 and 2
We followed protocol to make LB Agar Kan +Suc plates and LB broth.
 * 6/13/14: **

Plasmid: YopH Resistance: Kan Cell type: BL21 (DE3) cells
 * 6/12/14**
 * Transformation efficiency**

We had 3 tubes; A, B and C. In A (1 ng) we put 4.55 ul of 1:100 diluted stock plasmid. In B (5 ng) we put 2.27 ul of 1:10 diluted stock plasmid. In C (25 ng) we put 1.14 ul of undiluted stock plasmid. Each tube had varying amount of plasmid in order to assess the transformation efficiency. The transformation efficiency is = # colonies/ng of plasmid. Overall only place C showed significant growth of colonies after incubation with a transformation efficiency of 5.6. See results pictured below.



**Days 1 &2:** Computer Lab: We started by going through all of the Spring 14 Target Discovery pages, verified that the information was all correct and chose which ones we decided would be "kept" and which would be "trash." In addition to these we screened a list of unknown targets called "Seattles," found the required information for each and then input all data into the Google doc. Again decided which ones we wanted to keep and which ones to trash.

Follow protocol to submit DNA sequence to facility, Follow by nanodrop to determine concentration of plasmids.
 * 6/10/14**
 * Submitting to DNA sequence facility**



code >NW-T7 1045 256 3 0.05 GNNNNNNNNNNNNNNNNNNNNNNNNNNNNGTNANNNNTGGNNNAANGGCTGNGNNCNNGGNNCTTTNNGGGGGCCNCNNGGNNCNNANGNNNNNNNNNNCGCGNNNNACCCNACNGCCCCGNNNTNNNNNTCNNNNNNNNCCCTCNNGGGNACCTCNNTGGAAAGGAAGCGGGANAAGGGGGCNACCGTGTTCNCAAACGTNNCANGTGATTCCTTCNNNNNCAGAATGANCCNNANNACATGATGAAAAAANAGAAAAAGTCTGAAGCTCTGAATANNNAGGAGTACTTTTGCATGTGATGGGANNTGCCCTGGGGTCCGTGGTTGTGGTCATCACGGCCATCATATTCTATGTGCTTCCCCTGAAGAGTGANGACCCGTGTAACTGGNGNTGTTACATTGACCCCAGCCTGNCTGTCCTNNTGGNCNTCATCATTTTGTCNTCTGCCTTCCCGCTTATCAAGGANANNNNTGCNNTTCTGCTACNNATGGTCCNNNANGGAGTCAACATGGAAGAGCTGATGAGTAAACTCTCTGCTGTGCCTGGAATTAGCAGTGTACATGAANTGCACATCTGGGAACTTGTAAGTGGAAANATTATTGCCNCNCTGCNNATCANGTATCCTAAGGACAGGGGATATNNNNATGCCNNCACAAAAATTCGAGAAATCTTCCACNATGCGGGAATCCNNNNTGTGACCATCCNGTTTGANNNTGTGGACNTTGAANGAACCCCTGGANCANANNACTTACTGTTGCTCTGCANCTCANCCTGNATCTCCNAGGGCTGTGCTANGNNNTGTGTTGTCCCCCCGGGGCACTGCCTCTGGCTCACGNCAATGGCTGTGCTNANCNCNATGGTGGGCCCTCNCTAGACNNNTNNNNAAGTGATNGGCCTCNNTANANNANANGCCANANAATGGCTANTGNANTGTCTTTGGATAGCTGNNTAATGACCNNGGNNAANNCTTAACAANNTNNNGANGNNCNNTGNTATGTCAACNNNNNNATTTTNNNGGTTAAGGGCGAATTCCNNCNNNCNGNN code Above are the results from DNA sequencing facility. Protein is pmcherry; backbone is pGEMt and primers used are sp6 (forward) and T7 (reverse).
 * Results from DNA sequencing: **
 * T7 terminal:** GCTAGTTATTGCTCAGCGG
 * SP6 promoter:** ATTTAGGTGACACTATAG

Purpose of this was to dilute primers that we had ordered for cloning. Pictured above is the Integrated DNA Technologies Specification Sheet. Included in sheet are the pLIC-rev sequence, properties and amount of oligo.
 * 6/6/14**
 * Primer Dilution**
 * Order number:** 10439966
 * Reference number:** 122155112C