Andrew+B. 


 * January 19, 2013**

Protein expression day. Two methods were attempted. 2 x 500ml cultures were grown up using started cultures grown overnight in 37C incubator to an OD of 0.6. They were then induced and overexpressed for 5 hours. 1 x 1000ml culture was grown up from a plate colony in 37C incubator until it reached an OD of 0.6. This was then induced and transfered to a RT shaker for 20hr before centrifugation.

Andrew - show some of your scores - Dr. B 11/19/12
 * Virtual Screening Progress:**

Libraries Screened: - cb_306_3d.sdf - Fragment-set_3D.sdf - HF9_180_Plates_1um3D_catnum.sdf - NIH_ClinCol3Ded.sdf - ChemBridge-diversity3D.sdf - MayBridge50k_3D.sdf - MW-set_3D.sdf

In Progress:


 * Freebie from Journal Club**


 * November 29, 2012**

The SDS-PAGE gel from a few days ago was finally dried. The result is shown below. The band corresponding to PfDXR is at around 47kDa. Other contamination is seen that was hopefully eliminated in FPLC.



Lane 1: Fermentas PageRuler Pre-Stained Molecular Weight Standard Lane 2: Sample 0: Large cultures pre-induction at OD600 of 0.6 Lane 3: Sample 1: Large cultures post-induction Lane 4: Sample 2: Filtered Cell Lysate Lane 5: Sample 3: Flow-Through Lane 5: Sample 4: Wash Lane 6: Sample 5: Elution 1 (Band seen at around 47kDA) Lane 7: Sample 6: Elution 2


 * November 28, 2012**

Lots of aliquots were made for both NADPH and DXP, the cofactor and substrate respectively. NADPH was solubilized in 0.01M NaOH and diluted to 5mM. This gave us 192 aliquots at 5mM of 125uL (enough for 5 enzyme assays each assuming it retains functionality). Aliquoting was performed under minimal lighting due to the light-sensitivity of NADPH. DXP was solubilized in nanopure water and dilted to 12mM. This gave us approximately 165 aliquots at 12mM of 10uL (enough for 1 enzyme assay each). 50mL Tris-HCl at 125mM for assay was made from 500mM stock solution. First run enzyme assays will be attempted Friday evening.


 * November 27, 2012**

Harvested the cultures at around 8:45am via centrifugation. We then proceeded to protein purification with 1L worth of cell pellets. The remaining liter was left in -80C freezer in case something goes wrong. The cells were then sonicated and the protein purified via column chromatography with a nickel resin. An SDS-PAGE gel was run on samples taken throughout this process to characterize the protein and a band was seen at the right length in elution 1. Therefore, we decided to proceed with FPLC to further purify the protein after concentrating down elution 1 to 1mL. The FPLC result is shown below. There is one large peak at the appropriate size for a homodimer of PfDXR so the sample corresponding to that peak (25-29) were collected. They were then concentrated again down to 1mL. 500mL of this was snap frozen in liquid nitrogen and stored in -80C. Glycerol was added to the other half of the protein to 20% and then stored in -20C. Concentrations of both samples were taken (before snap freezing and after adding glycerol). For some reason, the concentrations were the same even though adding the glycerol should have decreased the concentration of that half. Maybe, the glycerol hadn't fully mixed throughout the solution although I pipetted it for a minute or so to mix it before taking that reading.

// Elution 1 of PfDXR Concentrated to 1mL Before FPLC //

A = 0.235 e = 39162.5 M-1 cm-1 b = 1 cm

c = A/Eb = (0.235) / (39162.5 M-1 cm-1) * (1 cm) = 6 microMolar. 6 microMolar = (6 micro moles / 1 liter) * (47206.6 g/mole) = 0.283 mg/ml yield = 0.283 mg

//FPLC Graph - 1// PfDXR homodimer is present in fraction samples 25 through 29. Presence can be verified by molecular standards (77 kDa is the small bump near fraction samples 29 through 31). PfDXR monomer is approx. 47 kDa (dimer will be approx. 94 kDa).

//FPLC Graph - 2 (Rescaled to better show the peaks)// Small bump at fractions 31 through 34 may indicate monomer - standard at fractions corresponding to 33 through 36 is approx. 33 kDa.

//PfDXR Concentrated to 1mL After FPLC (0.5mL in 20% Glycerol)// A = 0.291 e = 39162.5 M-1 cm-1 b = 1 cm

c = A/Eb = (0.291) / (39162.5 M-1 cm-1) * (1 cm) = 7.43 microMolar. 7.43 microMolar = (7.43 micro moles / 1 liter) * (47206.6 g/mole) = 0.351 mg/ml yield: 0.1755 mg

//PfDXR Concentrated to 1mL After FPLC (0.5mL snap-frozen)// A = 0.294 e = 39162.5 M-1 cm-1 b = 1 cm

c = A/Eb = (0.294) / (39162.5 M-1 cm-1) * (1 cm) = 7.51 microMolar. 7.51 microMolar = (7.51 micro moles / 1 liter) * (47206.6 g/mole) = 0.354 mg/ml yield: 0.177 mg

total yield after FPLC: 0.354 mg


 * November 26, 2012**

Started 2L of expression. Each large culture was "started" with 50mL of starter culture. They were grown at 37C until an OD600 of around 0.6 was reached. They were then induced with IPTG and moved to room temperature shakers for a 20h growth. Will spin down and purify tomorrow.


 * November 25, 2012**

Started overnight starter cultures for expression on the 26th. Grew two flasks of 100mL each in anticipation of growing 2L of large culture.

112612 - Good. Dr B Transformation was successful for both colonies. Spun down cultures and Midiprepped them. The results from the second set of four pellets is shown below.
 * November 21, 2012**

//Nanodrop of PfDXR after Midiprep//


 * November 20, 2012**

No mutations were found in all of the colonies sent for sequencing. We went ahead and transformed colonies 5 and 15 into BL21 (DE3) competent cells. Also, we started a 320mL culture of colony 5 for Midiprep. Will spin down and Midiprep tomorrow.


 * November 18, 2012**

Spun down colonies and Kaarthik and I Miniprepped them. They all showed concentrations above 50 ng/ul. We then did a RE digest with HincII resulting in the gel images below. Colonies 5, 6, 15, 16, 21, and 22 will be sent to sequencing Monday in hopes of finding a colony without significant mutations that can be used for expression.

//Gel Image of RE Digest of PfDXR in pNIC-Bsa4// //with HincII for Colonies 1-8// Lane 1: Skip Lane 2: 1kb Ladder Lane 3: Colony 1 Lane 4: Colony 2 Lane 5: Colony 3 Lane 6: Colony 4 Lane 7: Colony 5 Lane 8: Colony 6 Lane 9: Colony 7 Lane 10: Colony 8

//Gel Image of RE Digest of PfDXR in pNIC-Bsa4// //with HincII for Colonies 9-16// Lane 1: Skip Lane 2: 1kb Ladder Lane 3: Colony 9 Lane 4: Colony 10 Lane 5: Colony 11 Lane 6: Colony 12 Lane 7: Colony 13 Lane 8: Colony 14 Lane 9: Colony 15 Lane 10: Colony 16

//Gel Image of RE Digest of PfDXR in pNIC-Bsa4// //with HincII for Colonies 17-24// Lane 1: Skip Lane 2: 1 kb Ladder Lane 3: Colony 17 Lane 4: Colony 18 Lane 5: Colony 19 Lane 6: Colony 20 Lane 7: Colony 21 Lane 8: Colony 22 Lane 9: Colony 23 Lane 10: Colony 24


 * November 17, 2012**

I checked the plates around 10:30am and most showed some colonies. I came back around 3:30pm and grew up small cultures with Kaarthik. We decided to grow up 24 colonies for Miniprep to increase chances of finding a positive clone without mutations. The growth was started at around 4:00pm. We will come back at 10:00am tomorrow to spin down and Miniprep. If time permits an RE digest will be done to check for positive clones.


 * November 16, 2012**

I performed a DPNI digest of the plasmid PCR product. The nanodrop of the result is below. I then proceeded on to cloning. I did a digestion of pNIC-Bsa4, cohesive end generation, annealing and transformation, and plated around 7:45pm. Will check for colony growth tomorrow morning.

//Gel Image of Digest of pNIC-Bsa4 with BsaI// Lane 1: Skip Lane 2: 1kb ladder Lane 3: pNIC-Bsa4 Digest with BsaI Lane 4: Skip Lane 5: pNIC-Bsa4 Digest with BsaI Lane 6-10: Skip

//Nanodrop of Modified PfDXR After DPNI Digest and Clean up//


 * November 8, 2012**

I was going to attempt a complete restart of the project, making a new oligo mix including just the primers needed for the modified insert. In trying to determine how many of the original primers to omit, we noticed that the custom primers we designed for modifying the insert were incorrect. They had been created based off of the unmodified CDS rather than the optimized CDS. This explains the terrible PCR results. The forward primer was redesigned with the sequence below. This should allow for successful PCR.

5'-­ TAC TTC CAA TCC ATG AAA AAG CCT ATC AAC GTA -­3'


 * November 7, 2012**

Group attempt at PCR off plasmid DNA and oligio primer DNA. Unsuccessful results all around.

Lane 1: 1kB DNA Ladder Lane 2-5: Alex's vain attempt using modified Aptamer PCR protocol on plasmid DNA (15 cycles) Lane 6: Kaarthik's vain attempt using custom protocol on plasmid DNA (30 cycles) Lane 7: Kaarthik's vain attempt using primary mix DNA with custom protocol. (30 cycles) Lane 8: Andrew's vain attempt using Aptamer protocol (no modifications) on plasmid DNA (15 cycles) Lane 9-12: Recheck of secondary PCR with new primers (20 cycles only).


 * November 5, 2012**

Attempted a PCR off the plasmid DNA for the positive clone that we are trying to copy off of to truncate the protein. No results were seen on the gel. Perhaps thermocycling conditions or concentrations of reagents/starting plasmid will need to be altered some more.


 * November 3, 2012**

The PDF below shows a visual comparison between the top six ranked GOLD ligands and their corresponding docking in ICM. A table is included at the bottom that gives the GOLD and ICM scores for each ligand.




 * November 2, 2012**

I prepped the protein samples collected during expression and purification for SDS-PAGE analysis. I ran the PAGE using a pre bought gel and then stained it. Alex or Kaarthik will destain it and dry it tomorrow.


 * November 1, 2012**

Kaarthik sonicated the cell cultures. I spun them down about 2 hours later. Proceeded to purification and completed column chromatography. Will run the protein gel tomorrow.


 * October 29, 2012**

Ran some analysis on the virtual screening results. Modified the output files with SED and ran the statistical analysis in R. Below in a graph of the GOLD vs ICM scores for the top ligands from the secondary GOLD screen. The blue boxes represent the ligands that scored higher than 70 in the GOLD run and the blue line represents the linear model created for this set. The red line represents the linear model created for the total set of ligands.


 * October 26, 2012**

Expressed protein in 2 separate 500mL cultures. Each was grown with 1% glucose in LB to hopefully prevent any "leaky" translation that could cause issues for toxic proteins. Expressed for the full 4 hours at 37C. Will proceed to sonication and purification next week.

Grew starter cultures for protein expression tomorrow.
 * October 25, 2012**

102112 - Andrew - ok looks like it is workable, especially if just using for PCR - Dr. B
 * October 20, 2012**

Took plates out of the incubator and all of them had grown. The transformation plates had about 40 or so colonies on each plate. There was no distinguishable difference between plate A or plate B so either amount of plasmid should work fine for future transformation. I spun down the large cultures and midiprepped one culture. Kaarthik midiprepped the other. When transferring eluted DNA and isopropanol to final syringe, a large of amount of sample was lost due to an unfortunate turn of events. However, I lowered the final eluting volume to 250uL to try to account for this loss of DNA and still get a workable concentration. This seemed to work as I got a concentration of 74ng/uL.

//Nanodrop of Midiprepped PfDXR DNA//


 * October 19, 2012**

Continued screening libraries. Transformed the miniprepped positive clone colony into BL21 competent cells. Plate A was transformed using 0.75uL of plasmid and Plate B was transformed using 0.5uL plasmid. I also made a backup master plate for the positive clone. Placed in incubator for overnight growth. Started a large growth culture of dH5alpha from the positive clone for midiprep on Saturday.


 * October 18, 2012**

New target acquired (PfDXR) in collaboration with Kaarthik. Started virtual screening runs against the existing crystal structure with the HF9 libraries.

101612 - Andrew, ok good. I am interested to see how the regularized homology model compares to the un-regularized model. - Dr. B
 * October 11, 2012**

Started working through the virtual screening protocol for my target. I was unable to get the Hermes visualizer to work properly when trying to define my active site manually by picking an area around a molecule in the active site. Since there was no ligand in my model file, I decided to align it with the known PDB structure that it was based off of (1VSV chain B) giving an RMS of 0.062. I then deleted everything from the PDB structure except the ligand crystallized in 1VSV and saved a new molecule with the ligand still aligned against model. It seemed to fit very well in what looked to be the active site so I used this as my ligand in the gold configuration set up. This first screen that I am running is against the HF9 library using gold. In the future, I will also screen against this library using ICM and will try the regularization in ICM and compare results.


 * October 10, 2012**

I cleaned up and nanodropped my gel extraction product. The concentration was disappointingly low for the amount of sample that was run through the column. Perhaps the gel extraction column was overloaded, resulting in loss of sample.

//Nanodrop of PCR Product After Gel Extraction and PCR Cleanup//


 * October 9, 2012**

Ran a gel extraction on the PCR2 product made earlier in the week. I didn't get a chance to clean it up or check concentrations. Completed the Homology Modeling protocol via the SWISS MODELING sever. Template Identification and Automated mode modeled my protein off of two different proteins. I ended up going with the one with that the template identifier found as my template (1VSV chain B) for modeling because it had a better QMEAN Z-score and a better QMEAN4 score. It also matched up with one of the chains that BLAST identified as having the highest identity and score. If screening yields poor results with this model, I will try the alternative identified by the automated mode alone (3CIF chain D). I aligned the two structures in PyMOL and they aligned pretty well with an RMS of 0.329 so at least they are fairly similar meaning that their searching algorithms are pretty consistent. I also aligned the model based of off 1VSV B with 1VSV chain B and it aligned with an RMS of 0.062, a very good score. This was expected though since 1VSV was used as the template. I ran my model through the Mol Probity website and got pretty bad results for all the parameters tested. However, this was expected as it was built off of a model with only 34% identity. I will start virtual screening against the HF9 library soon.

//Gel Image of PCR Squared Prior To Gel Extraction// Lane 1: 1kb Ladder Lane 2-12: PCR Squared Product


 * October 8, 2012**

No growth seen on plates. Made a bunch of PCR2 product in anticipation of a future gel extraction.

100912 - Andrew - ok, we also have new T4 DNA Polymerase. that you could try on the next round. - DR. B I started cloning again today. I did cohesive end generation (using the new dCTP/dGTP) and annealing and transformation. The two concentrations that I tried out were 2 accepting vector, 4 PCR insert and 4 accepting vector and 6 PCR insert. I plated around 5:45pm.
 * October 6, 2012**


 * October 5, 2012**

I digested pNIC-Bsa4 successfully this time and got higher concentrations that should be high enough to work. Also, I ran a gel to check that it actually cut and both digests were successful.

//Nanodrop of pNIC-Bsa4 After Digest with BsaI - 1//

//Nanodrop of pNIC-Bsa4 After Digest with BsaI - 2//

//Gel Image of pNIC-Bsa4 Cut with BsaI// Lane 1: Skip Lane 2: 1kb Ladder Lane 3: pNIC-Bsa4 cut with BsaI - 1 Lane 4: pNIC-Bsa4 cut with BsaI - 2 Lane 5-10: Skip


 * October 4, 2012**

I Midi-prepped pNIC-Bsa4 pellets that I grew up last week. I eluted in 500uL of 10mM Tris-HCl and got a pretty good concentration of around 106ng/uL and decent purity ratios. I will send to sequencing on Monday to ensure that no mutations were introduced at critical sites. I also attempted a digest of pNIC-Bsa4 with BsaI but failed to get good enough concentrations to feel comfortable with proceeding. I will attempt the digest again tomorrow and proceed with cloning on Saturday.

//Nanodrop of pNIC-Bsa4 Plasmid after Midiprep -Trial 1//

//Nanodrop of pNIC-Bsa4 Plasmid after Midiprep - Trial 2//

//Nanodrop of Digest of pNIC-Bsa4 with BsaI//


 * October 1, 2012**

Checked plates and observed no growth. Images of the plates are displayed below. Will proceed to try cloning again. At least we know the antibiotic on these plates is still effective.

//Image of Plate A - no colonies observed// //Image of Plate B - no colonies observed//


 * September 29, 2012**

Spun down pNIC culture to harvest pellet and stored it in -20C freezer. I will midiprep the pellets next week at some point. I also started cloning and did cohesive end generation followed by annealing and transformation. I plated at around 2:00pm and will check for colonies tomorrow in the late afternoon. For my tube B, I tried using 10uL of my insert and 4uL of the accepting vector.


 * September 28, 2012**

I prepared my pNIC-Bsa4 vector for cloning by cutting with BsaI for three hours in the 37C water bath. I ran a gel to confirm it cut and then combined my two samples into one cleanup column to hopefully increase final concentration. I then used the nanodrop to determine concentration (results below). It appears to have cut properly and the concentration and purity ratios seem to be good enough to proceed. Also started a 160mL culture from an already made plate of dH5alphas transformed with pNIC-Bsa4. This will hopefully replenish my dwindling stock of pNIC-Bsa4.

//Gel Image of pNIC-Bsa4 Cut with BsaI// Lane 1: Skip Lane 2: 1kb Ladder Lane 3: Cut pNIC-Bsa4 -1 (AB) Lane 4: Cut pNIC-Bsa4 -2 (AB) Lane 5-6: Skip Lane 7: 100bp Ladder Lane 8-9: UM Samples Lane 10: Skip

//Nanodrop of Cut pNIC-Bsa4 After Clean Up//


 * September 26, 2012**

I ran my gel extracted sample through PCR cleanup and used the nanodrop to determine concentration. The concentration was a little lower than hoped for but at least it is known to be pure. The 260/230 value improve drastically from the previous step to an excellent value and the 260/280 value is good as well. Updated lab notebook. Will procede with cloning as soon as is possible.

//Nanodrop Result After PCR Clean Up//


 * September 25, 2012**

Performed a gel extraction of my PCR squared product from yesterday. An image of the gel prior to extraction is shown below. I used a new gel extraction kit which yielded interesting results. I got out a decent concentration of product (eluted in 50uL Tris-HCL) of around 58 ng/uL. The 260/280 values also looked to be fairly good. However, the 260/230 values seemed extremely low although I'm not sure exactly what to expect at this stage as it has not yet been cleaned up fully. I did a gel check of the gel extracted product (ran 3uL) and it looked pretty good and verified that the concentration was decent. Perhaps there are still some unwanted salts in solution with the DNA and running it through our other cleanup kit would solve that issue (I think this is what we did in the summer). Since I was testing a new product though, I wanted to get as much data on my results as possible.

//Gel Image of PCR Squared Product Prior to Extraction// Lane 1: 1kb Ladder Lane 2-12: PCR Squared Product

//Nanodrop Result After Gel Extraction Kit (Trial 1)//

//Nanodrop Result After Gel Extraction Kit (Trial 2)//

//Gel Image of PCR Product After Gel Extraction//


 * September 24, 2012**

Failed attempt at gel extraction due to mistake with gel. Re-did secondary PCR and PCR squared for a gel extraction Tuesday. The secondary PCR results looked good and the appropriate size (approx 1.1kb) so hopefully the PCR squared will turn out good as well.

//Gel Image of Two Secondary PCR Reactions// Lane 1: Skip Lane 2: 1kb Ladder Lane 3: Secondary PCR - 1 Lane 4: Secondary PCR - 2 Lane 5-12: Skip

Andrew -- good deal. Include a 'result' - e.g. snapshot of your best ranking list form Virtual or etc. -- Dr. B Worked on virtual screening refresher. Performed PCR squared with the intention of gel extracting and cloning on Monday (made 600uL). Made 4L of LB for Joey's current project as well.
 * September 20, 2012**

Virtual Screening Results


 * September 19, 2012**

Received sequencing results from the DNA core. No positive clones were identified. All samples came back showing the presence of the pNIC-Bsa4 vector without the SacB gene. However, it did not have my gene of interest in its place. Below is an example of the pairwise alignment I did using BLAST comparing the CDS of my gene to the sequencing results. All results were fairly similar to this with only a little similarity on the end of the read. Since this round of cloning failed, I will start another as soon as possible (probably Monday as I won't be able to come in on the weekend). The plan for the rest of the week is to work on the virtual screening refresher and if lab time is still needed, make LB/other stocks that are running low.




 * September 17, 2012**

Prepared samples for DNA sequencing and sent off the 12 samples with pLIC forward primer at around 3:00pm. If a sample looks to contain a positive clone, then the pLIC reverse primer will also be sent to ensure integrity of the clone.


 * September 15, 2012**

Miniprepped the pellets from yesterday and determined concentration of each using nanodrop. The 260/230 values are a little lower than preferred but we will see what happens. The concentrations seem decent but as seen from previous cloning trials, not indicative of success.

Concentrations after Miniprep of Colonies 11 and 12

Concentrations after Miniprep of Colonies 9 and 10

Concentrations after Miniprep of Colonies 7 and 8

Concentrations after Miniprep of Colonies 5 and 6

Concentrations after Miniprep of Colonies 3 and 4

Concentrations after Miniprep of Colonies 1 and 2


 * September 14, 2012**

Spun down small cultures to pellet cells. Saved in -20C freezer.


 * September 13, 2012**

Checked cultures and they weren't overgrown (maybe about 25-35 colonies). Made a master plate with twelve of them and started small cultures of those twelve colonies for miniprep.


 * September 12, 2012**

Did cohesive end generation and transformation and annealing today. Plated the cultures at 10:30pm.


 * September 11, 2012**

Prepared my pNIC-Bsa4 accepting vector by digestion with BsaI. Ran digested vector through cleanup kit and determined concentration using nanodrop. Ran 3uL on a gel to check that it cut properly.

Nanodrop of cut pNIC-Bsa4

Lane 1: Skip Lane 2: 1kb Ladder Lane 3: RE Digest of pNIC-Bsa4 with BsaI (AB) Lane 5-6: RE Digest of pNIC-Bsa4 with BsaI (KR) Lane 7-10: Skip


 * September 10, 2012**

Ran PCR Squared product through the PCR clean up kit. Decided not to gel extract. Measured concentration using nanodrop. Nanodrop of tcGAPDH


 * September 8, 2012**

Did PCR Squared using Q5 polymerase. The lanes aren't as clean as I'd like so I'm considering doing a gel extraction next week before trying to clone with it. Lane 1: Skip Lane 2: 1kb Ladder Lane 3-6: PCR Squared Lane 7-10: Skip

Did primary and secondary PCR using new Q5 polymerase. It seems to have worked just as well as KOD. I used the primers designed for insertion directly into pNIC-Bsa4. Lane 1: Skip Lane 2: 1kb Ladder Lane 3: Primary PCR Lane 4: Secondary PCR (approx. 1.1kb) Lane 5-10: Skip
 * September 7, 2012**

Did PCR squared and used all of remaining good secondary (800uL of PCR squared) for gel extract. Gel extracted and ran through kit. Simultaneously cut pNIC-Bsa4 accepting vector with BsaI for use in transformation.
 * August 1, 2012**

PCR Squared - 1 Lane 1: Skip Lane 2: 1 kb Ladder Lane 3-11: PCR Squared

PCR Squared - 2 Lane 1: Skip Lane 2: 1 kb Ladder Lane 3-11: PCR Squared

Did primary and secondary PCR again (used polymerase B). Got two secondaries that look good, will proceed with PCR squared and gel extraction tomorrow.
 * July 31, 2012**

Lane 1: Skip Lane 2: 1kb Ladder Lane 3: Primary PCR -1 Lane 4: Secondary PCR - 1 Lane 5: Secondary PCR -1 Lane 6: Primary PCR - 2 Lane 7: Secondary PCR - 2 Lane 8: Secondary PCR -2 Lane 9: Primary PCR - 3 Lane 10: Secondary PCR - 3 Lane 11: Secondary PCR -3

Sent samples to sequencing. Remade Oligo mix and attempted primary and secondary PCR. Checked gel and saw nothing (no picture). Realized that I had used a polymerase that was bad according to Larry's tests earlier this summer (polymerase E). Will try PCR again tomorrow.
 * July 30, 2012**

Spun down samples and miniprepped. Concentrations obtained via Nanodrop (see below). Didn't have time to send to sequencing (will do on Monday). Remembered to take master plate out of incubator this time.
 * July 27, 2012**

Nanodrop of Colony 11 and Colony 12

Nanodrop of Colony 9 and Colony 10

Nanodrop of Colony 7 and Colony 8

Nanodrop of Colony 5 and Colony 6

Nanodrop of Colony 3 and Colony 4

Nanodrop of Colony 1 and Colony 2

Checked plates from previous day's transformation. Plates seem to have a lot of colonies (in the hundreds) which is suspect, might get empty colonies again. Proceeded to grow up 12 colonies for miniprep and made master plate. Made LB+AMP plates in preparation for PUC19 cloning.
 * July 26, 2012**

Got sequencing results back but they were empty colonies. Only containing genomic DNA (sequence results only showed "NNNN" for most sent samples). Proceeded with transformation and annealing from previously cut pNIC-Bsa4 and gel extracted plasmid. If this doesn't give a positive clone, will try cloning into PUC19.
 * July 25, 2012**

Looked at Homology Modeling Protocols. Realized that there were two forms of my protein target in my organism (a cytosolic and glycosomal form). Currently working on cloning the cytosolic form, might want to look for inhibitors for both forms and eventually clone/express both proteins.
 * July 24, 2012**


 * July 23, 2012**

Miniprepped 12 samples and sent them for DNA sequencing using the pLIC forward primer. Began to look at virtual screening protocols.

Measured concentrations of Miniprepped samples using the Nanodrop Spectrophotometer Nanodrop of Colony 11 and Colony 12

Nanodrop of Colony 9 and Colony 10

Nanodrop of Colony 7 and Colony 8

Nanodrop of Colony 5 and Colony 6

Nanodrop of Colony 3 and Colony 4

Nanodrop of Colony 1 and Colony 2


 * July 21, 2012**

Spun down twelve samples after 16 hours of growth in 37 degree shaker. All samples formed pellets. Stored in -20 freezer. Forgot to take master plate out of incubator.


 * July 20, 2012**

Checked transformed plates and observed colony growth. Proceeded to make master plate and grow up colonies (small scale) for Miniprep. Chose 12 colonies in hopes of increasing chances of finding one with a successful insert.

Nanodrop of Cut pNIC-Bsa4 07/19/12

Gel Check of Cut pNIC-Bsa4 07/19/12 Lane 1: Skip Lane 2: 1 kb ladder Lane 3: RE Digest of pNIC-Bsa4 with BsaI (AB) - 1 Lane 4: RE Digest of pNIC-Bsa4 with BsaI (AB) - 2 Lane 5: Skip Lane 6: Secondary PCR (KR)

Nanodrop of Cut pNIC-Bsa4 07/18/12 -NOTE: Forgot the incubation step during cleanup which is probably why the yield is so bad.

Gel Check of Cut pNIC-Bsa4 07/18/12 Lane 1: Skip Lane 2: 1 kb Ladder Lane 3: RE Digest of pNIC-Bsa4 with BsaI (AB) Lane 4: RE Digest of pNIC-Bsa4 with BsaI (YH) Lane 5: Skip Lane 6: PCR Clean Up (KR) Lane 7: PCR Clean Up (KR) Lane 8: RE Digest of pNIC-Bsa4 with BsaI (KR) Lane 9: pFAL (KR) Lane 10: Skip

Nanodrop of tcGAPDH after PCR Clean Up on the Two Gel Extraction Products Below 7/17/12

Nanodrop of Column 2 After Gel Extraction 7/17/12

Nanodrop of Column 1 After Gel Extraction 7/17/12

PCR Squared Before Gel Extraction 07/17/12 Lane 1: Skip Lane 2: 1 kb Ladder Lane 3-11: PCR Squared of tcGAPDH (approx. 40uL in each well) Lane 12: Skip

Nanodrop of Gel Extract 07/17/12 Trials 1 and 2 respectively of Gel Extract Attempt for Column 2

Nanodrop of Gel Extract 07/17/12 Trials 1 and 2 respectively of Gel Extract Attempt for Column 1

PCR Squared Before Gel Extraction 07/17/12 Lane 1: Skip Lane 2: 1 kb Ladder Lane 3-11: PCR Squared of tcGAPDH (approx. 40uL in each well) Lane 12: Skip

Secondary PCR 07/17/17 Lane 1: Skip Lane 2: 1 kb ladder Lane 3: Secondary PCR 1 Lane 4: Secondary PCR 2

Received sequencing results of PCR products which validated the integrity of my oligo mix and primary PCR from 6/22/12. This primary PCR product was used to create these two secondary PCRs.

Overlap Assembly Attempt 07/16/17 Lane 1: Ski Lane 2: 1kb ladder Lane 3: Primary PCR (AB) Lane 4: Secondary PCR (AB) Lane 5: Secondary PCR (YH) Lane 6: Secondary PCR (YH)

SDS Page Gel of ftHAP 07/13/12 Lane 1: Protein Ladder Lane 2: Sample 0 Lane 3: Sample 1 Lane 4: Sample 2 Lane 5 Sample 3 Lane 6: Sample 4 Lane 7: Sample 5 Lane 8: Sample 6

Nanodrops of Elution 1 and 2 for Two Separate Trials of Growing ftHAP 7/6/12 Elution 1 and 2 Respectively of ftHAP Grown up on 7/3

Elution 1 and 2 Respectively of ftHAP Grown up on 6/27

RE Digest of tcGAPDH in pNIC-Bsa4 with PvuII 07/09/12 Lane 1: 1kb ladder Lane 2: Uncut pNIC-Bsa4 Lane 3: Control (pNIC-Bsa4 without insert cut with PvuII) Lane 4: Sample 1 Lane 5: Sample 2 Lane 6: Sample 3 Lane 7: Sample 4 Lane 8: Sample 5 Lane 9-10: Skip (Not enough sample left) Lane 11: Sample 8

RE Digest of tcGAPDH in pNIC-Bsa4 with PvuII 07/09/12 Lane 1: 1kb ladder Lane 2: Uncut pNIC-Bsa4 Lane 3: Control (pNIC-Bsa4 without insert cut with PvuII) Lane 4: Sample 1 Lane 5: Sample 2 Lane 6: Sample 3 Lane 7: Sample 4 Lane 8: Sample 6 Lane 9: Sample 7 Lane 10: Sample 5

Virtual Digest of pNIC-Bsa4 with PvuII With and Without tcGAPDH Insert (1kb ladder) Samples Sent to DNA Core for Sequencing 7/6/12

Nanodrop of Colony 8 (Trial 1 and 2): Average Concentration of 29.15 ng/ul

Nanodrop of Colony 7 (Trial 1 and 2): Average Concentration of 24.95 ng/ul

Nanodrop of Colony 6 (Trial 1 and 2): Average Concentration of 35.1 ng/ul

Nanodrop of Colony 5 (Trial 1 and 2): Average Concentration of 18.15 ng/ul

Nanodrop of Colony 4 (Trial 1 and 2): Average Concentration of 29.9 ng/ul

Nanodrop of Colony 3 (Trial 1 and 2): Average Concentration of 20.45 ng/ul

Nanodrop of Colony 2 (Trial 1 and 2): Average Concentration of 28.6 ng/ul

Nanodrop of Colony 1 (Trial 1 and 2): Average Concentration of 22.25 ng/ul

RE Digest of pNIC-Bsa4 06/2912 Lane 1: Skip Lane 2: 100bp ladder Lane 3: RE Digest of pNIC-Bsa4 with BsaI (MT) Lane 4: RE Digest of pNIC-Bsa4 with BsaI (DO) Lane 5: RE Digest of pNIC-Bsa4 with BsaI (AB) Lane 6-10: Skip

Nanodrop of pNIC Bsa4 Accepting Vector 06/2912

070212 - good luck with Annealing and Transformation today. -- DR. B

Attempted annealing and transformation with the pNIC Bsa4 shown below but failed to get colony growth on my plates. Recut the accepting vector and got a higher concentration (see above).

Nanodrop of pNIC Bsa4 Accepting Vector 06/26/12 260/280: 2.12 260/230: 0.87

Gel Check of Digest of pNIC Bsa4 Accepting Vector 06/26/12 Lane 1: skip Lane 2: 1kb Ladder Lane 3: pNIC Bsa4 Digest with BsaI (AB) Lane 4: pNIC Bsa4 Digest with BsaI (LH)

Nanodrop of tcGAPDH 06/26/12 PCR Squared tcGAPDH 06/26/12 Lane 1: Skip Lane 2: 1kb Ladder Lane 3: PCR Squared A Lane 4: PCR Squared B

Lane 5: PCR Squared C Lane 6: PCR Squared D Lane 7-10: Skip

PCR pNIC-Bsa4 06/25/12 Primers: pLIC-for and pLIC-rev Lane 1: Skip Lane 2: 100 bp DNA ladder 5 ul Lane 3: ? ng pNIC-Bsa4 KRAB Lane 4: ? ng pNIC-Bsa4 KRAB Lane 5: Control KRAB Lane 6: Skip Lane 7: ? ng pNIC-Bsa4 AH Lane 8: ? ng pNIC-Bsa4 AH Lane 9: ? ng pNIC-Bsa4 AH Lane 10: Control AH

Sample No. 3 KRAB (smallest amount of pNIC-Bsa4) is not shown above because it was misplaced after being taken out of the PCR machine.

062112 - Andrew - add your gel images of PCRs. (in reverse) -- Thanks, Dr. B

PCR Overlap of //T. Cruzi// GAPDH 6/22/12 Lane 1: Skip Lane 2: 1kb Ladder Lane 3: 100bp Ladder Lane 4: Primary PCR Lane 5: Secondary PCR (Option A), (Approx. 1.1kb) Lane 6: Secondary PCR (Option B), (Approx. 1.1kb) Lane 7: Secondary PCR (Kaarthik R.) Lane 8-10: Skip

PCR Overlap of //T. Cruzi// GAPDH 6/21/12 Lane 4: 1kb Ladder Lane 5: Primary PCR Lane 6: Secondary PCR (Option A) Lane 7: Secondary PCR (Option B)

PCR Overlap 6/20/12 Lane 1: Skip Lane 2: Skip Lane 3: 1 kb DNA Ladder Lane 4: Primary PCR AB Lane 5: Secondary PCR (Option A) AB  Lane 6: Primary PCR KR  Lane 7: Secondary PCR (Option A) KR (Approx. 1.5 kb). Lane 8: Skip Lane 9: Skip Lane 10: Skip

06/13/12 PCR of pGBR22 using M13 Primers We repeated the experiment from ** June 07, 2012 ** but used the M13 Primer Set instead of the SP6/T7 Primer Set.

Lane 1: Skip Lane 2: 100bp DNA ladder Lane 3: 0.3 ng (1% conc.) pGBR22 plasmid KR Lane 4: 3 ng (1% conc.) pGBR22 plasmid KR  Lane 5: 30 ng (10% conc.) pGBR22 plasmid KR  Lane 6: No plasmid Lane 7: 0.3 ng (1% conc.) pGBR22 plasmid AB Lane 8: 3 ng (1% conc.) pGBR22 plasmid AB  Lane 9: 30 ng (10% conc.) pGBR22 plasmid AB  Lane 10: No plasmid. Maybe contamination from Lane 9.

Transformation of DH5alpha cells with pGFP 06/11/12: NOTE: PLATE A AND C WERE REVERSED Plate A: 25ng of Plasmid, 224 colonies/ng Plate B: 5ng of Plasmid, 800 colonies/ng Plate C: 1ng of Plasmid, 1200 colonies/ng

PCR of pGBR22 using SP6/T7 Primers 06/07/12 Lane 2: 100bp DNA ladder Lane3-10: PCR Samples of pGBR22 using SP6/T7 Primers

GoogleDoc [|Link] to Analyze DNA Sequence Results Document:

Restriction Digest Result Gel: 06/07/12 Lane 1: Skipped Lane 2: DNA Ladder Lane 3: Uncut Plasmid Lane 4: EcoRI Lane 5: PvuII Lane 6: EcoR1+ PvuII Lane 7: EcoRI KR Lane 8: PvuII KR Lane 9: EcoR1+ PvuII KR Lane 10: Uncut Plasmid KR

Nanodrop Trial 1: pGFP 1580ng/ul

Nanodrop Trial 2: pGFP 1580ng/ul