Aldo+D.

= Aldo's Research Page =

My cloning transformation plates for FrTu_DXR grew. I have three plates (taped together with blue tape) in the 4C refrigerator. I just wanted to let you know since you might also want to use them. I already picked some colonies & made my master plate. -Janice Kim

12/03/12: Dr. B, just a heads up, I'll be using the Wikispaces drop for the week of Thanksgiving.

- Need to purify protein and will characterize it soon.
 * Dec. 3:**
 * -** Continued to sonicate protein sample.

- after purification, the protein was stored
 * Dec. 4:**
 * -** purified protein using an old column. I used Joey's old FrTuHP column he used when he purified his protein.


 * Nov. 25-27**: was not able to come to lab

Nov. 28: - Worked on protein expression of Joey's FrTuHP. Prepared 2 500 ml flasks with 100 ml LB+KAN for growing up BL21(DE3) cells with FrTuHP CDS insert. - Will do the 12 hr expression overnight. Placed the 2 flasks in the shaking incubator at 8:30 PM today. - Will come back at 8 AM to continue through protocol tomorrow.

Nov. 29: - 8 AM: Only one flask had growth so I removed that flask from incubator and placed in 4 deg Cel. fridge for later use (3 hrs later) - I placed the other flask in the location where the flask that grew was at in the incubator, and let it grow for about 3 hrs. - Returned at 11 am and continued to grow up 2L flasks with LB+KAN until an initial OD600 of .1 was reached, then until an OD600 of .5 - Induced expression with IPTG and let grow for 4 hrs. - Spun down sample and stored in -80 deg. C freezer.

Nov. 30: not able to come to lab

112612 - ?? Dr B Aldo - include some results in your posts. - Dr. B 11/19/12
 * Week 11 - Thanksgiving Week: (Will use the Wikispaces drop on this one.)**
 * Week 10 (Nov. 12-16)**

Nov. 12: Had a test today, so not much work was done. Looked at Janice's cloned FrTu DXR colonies that she grew and they look okay. Will consider checking for positive clones.

Nov. 13: Decided to make a master plate of Janice's colonies from plate A of her samples. - Grew the bacteria for 16 hours in 37 C shaking incubator.

Nov. 14: Spun down the samples and stored in fridge. Will continue to check for positive clones when I have time. - Studied for an exam Thursday.

Nov. 15: Didn't come to lab; studied for exam


 * Week 9 (Nov. 5-9)**

Nov. 7: Worked on preparing another set of PCR insert and Accepting Vector for use in the annealing and transformation steps. - Will attempt cloning again once more, then, if it fails, possibly move on to another target given that the lack success has been prevalent in my target. - Attempted to grow up colonies onto another plate. Will check tomorrow.

Nov. 8: The plates have no growth yet. Usually at this point they will not grow at all. - Will check tomorrow as well.

Nov. 9: No growth on plates. - Continued to cut more pNIC plasmid for another round of transformation.


 * Week 8 (Oct. 29-Nov. 2)**

Oct. 29: Worked on cutting a pNIC vector for the next step. Lane 2: 1kb ladder Lane 4: cut pNIC plasmid

- Will use this cut plasmid for the Accepting Vector in the Annealing and Transforming steps that follow.

Oct. 30: Worked on preparing the PCR insert and the Accepting Vector for Annealing and Transformation. - Continued to prepare the solution to grow up colonies using DH5 alpha cells in 15 ml conical tubes in SOC media for 1hr. - Did a total of 2 plates (tube a and b) for growing up colonies overnight. - Worked on Virtual Drug Screening for my target - setting up for run 1

Oct. 31: Checked my plates - nothing has grown yet. - Decided to use my old plates from the Summer which did grow. I chose 8 colonies from plate A and made a master plate and attempted to grow those colonies in 15 ml conical tubes using 5ml of LB and Kan overnight in shaking incubator. I placed the master plate in the 37 deg C incubator. - There was only one more LB/Suc/Kan plate left which was used for my master plate. - I will need to make more for tomorrow if I attempt to retry growing the colonies. - Worked on Virtual Drug Screening - getting the results and compiling the results

Nov. 1: Checked my plates, and only 1 colony seemed to grow on plate A. No growth on plate B and no growth yet on my Master plate. Will need to get rid of those plates soon as they don't seem to work. - Decided to make more LB/Suc/Kan plates for future use.


 * Week 7 (Oct. 22-26):**

Oct. 22: - Worked on clean-up of my PCR squared sample and determined its concentration. - Finally a good concentration with good 260/280 and 260/230 values as well. This concentration is for my combined PCR squared samples after PCR cleanup. - Will continue to the next step soon when there is time.

102112 - Nice! Good work on getting it to work. Move on to cloning - but you may also need to upscale this 2 to 3 times as many samples if you don't get enough out of PCR cleanup. Also, if you want to team up with Janice and split the work some - you may be able to move faster. --Dr. B
 * Week 6 (Oct. 15-19):**

Oct. 19: - Worked on a PCR squared using Janice's secondary sample. Lane 1: skip Lane 2: 100 bp ladder Lane 3-6: stephanie and jennifer samples Lane 7-10: my PCR squared.

- Thanks to Janice, it seems like I finally got some PCR squared to work out fine that were the size of my gene. Hopefully after cleanup the concentrations are good enough to continue. - On a side note, it feels good to finally see this work, and that I used the old KOD polymerase that was labeled with markings all over indicating it was not good anymore (it seems it may still be working ok).

Another PCR secondary: Lane 1: skip Lane 2: 100bp ladder Lane 3&4: my secondary PCR

- Note that there was a bit of contamination on one of the bands. Hopefully this doesn't affect the PCR squared sample.

- Tried to do another one that is better but it didn't come out too good: Lane 2: 1 kb ladder Lane 3-7: others' samples Lane 8&9: my secondary PCR sample

- Note that the bands are there but are not too bright. - Will do another PCR secondary.

101612 - Aldo, try a few different annealing temps (57, 56 then 59 and 60) I assume your Primary PCR is working. However, if it isn't - then talk to Janice about using some of her primary to do your secondary. -- Dr. B - Lane 1: 100 bp ladder - Lane 2: my secondary PCR - rest of lanes: brandon and others' samples
 * Week 5:**
 * -** Worked on another secondary PCR reaction.

- Note that the gel image was did not look too good, but my sample still should of came out. - I will start another PCR primary mix, as the secondary completely failed. - Will check other annealing temps. for my target PCR procedure.

100912 - Aldo - show some gel images. We have nw T4 DNA Poly that may work better for you cloning (new dGTP and new dCTP also) - Dr. B Oct. 1-2: - Worked on cutting some pNIC-bsa4 plasmid
 * Week 4: Oct. 1-5**

10/3: - Worked on cutting more pNIC-bsa4 plasmid as the concentrations were not too good. - Continued to the cohesive end generation steps for both the PCR insert and accepting vector. - Continued to the Annealing and Transformation steps on the protocol, and will plate out the bacteria on Suc/Kan LB agar plates when acceptable. - Will do Master Plate Thursday (10/4/2012) if colonies grow.

10/4: - Colonies have not grown yet. Will wait another day to see if anything pops up. - In the meantime, another PCR squared reaction will be conducted in hopes of obtaining a higher concentration of my gene.


 * Week 3: Sept. 24 - 28**

Sept. 28: - Will cut pNIC-bsa4 plasmid in preparation for cohesive end generation. - Will do the next steps on Saturday as procedures require so at least a day after cutting the plasmid. - Lane 1: Max's cut pNIC vector - Lane 2: 1kb ladder - Lane 3: Aldo cut pNIC vector - Lane 4&5: Aldo other cut pNIC vector - Lane 7: 100bp ladder - Lane 8&9: Aldo PCR squared
 * -** Had 3 tests this week and did not have too much time to work on my research.

- Note that the cut vectors did have some contaminants. The two that look like they have contaminants are samples that were not cleaned up. The others look fine, though not too bright (probably low concentration). The two PCR squared samples look good too, and are the right size, although the bands could be darker.



- The concentration of the cut plasmid above is not too good after clean up.. will do it again.



- The concentration of the PCR squared sample is shown. Note that the concentration is not too good.


 * Sept. 18, 2012**

- Pooled together my previously gel-checked PCR squared products which did not show a dark band - which were good in terms of appropriate size - and gel-checked the combined samples:

Lane 1: skip Lane 2: 100bp ladder Lane 3-7: Janice's samples __Lane 9: ADO PCR squared__

- Note that the band finally looks good, with almost no visible contamination.

Concentration of PCR squared: ​



Average Concentration:
 * 372.25 ng/ul**

- Will continue to PCR cleanup soon.


 * Sept. 17, 2012**

- PCR squared results. Camera was taking bad pictures, I don't know why it was doing that.

Lane 1: skip Lane 2: 100bp ladder Lane 3-10: PCR squared samples from two different reactions (two attempts)

- A few came out good, most cannot be seen due to the image quality, but the bands showed the right size of the protein for all samples. - I will pool these samples together and run another gel-check to make sure they are good.


 * Sept. 14, 2012**
 * -** Ran another PCR squared reaction using previously made secondary PCR from the summer and from the recently made secondary.


 * Sept. 13, 2012**

PCR squared results: Lane 1: skip Lane 2: 100bp ladder Lane 4-7: PCR squared

- Note that there isn't too much PCR in my samples. The bands should be brighter, although they are the right size but just not enough to use. - Will do it again.


 * Sept. 12, 2012**

Secondary PCR:

Lane 1: skip Lane 2: 100 bp ladder Lane 3: secondary PCR sample.

- Size of Gene: **1158 bp**

- Results show a dim band at the correct size of the gene. I will continue to PCR squared, but will increase the amount of secondary PCR used for the reaction in hopes of obtaining a higher yield of my gene.

- Attempt at Primary PCR will be done. Possible gel results by today as well.
 * Sept. 10, 2012**

Gel Result: Lane 2: 1kb ladder Lane 3: Primary PCR, Aldo Lane 5&6: Janice Primary PCR

- Will continue to Secondary PCR by today.

- The procedures to clone my target into pNIC-Bsa4 will be done again. - PCR primary was completed using the new polymerase, but results showed that the gene did not amplify as shown in the gel. Lane 1: skip Lane 2: 1kb ladder Lane 3-6: karthik's samples Lane 9: my PCR primary sample
 * September 6, 2012**

- The gel shows that not all the genes were amplified as the band did not extend to the actual size of my gene. I used Block A on one of the PCR machines that is now labeled as "no good", so maybe that was the reason for the PCR failure.



__pNIC - bsa 4 Plasmid Cut__
July 20, 2012



Well 1: empty Well 2: 1kb ladder Well 3: Janice cut pNIC bsa4 Well 4: empty Well 5: Aldo cut pNIC bsa4

- pNIC-bsa4 cut plasmid was cleaned up using the PCR clean up procedure.
 * __July 9, 2012__**

//Nanodrop Results://

prepare another cut plasmid for future use if the previous fails.
 * Abs. of .195 is not too good. Regardless, I will continue with this sample throughout the protocol and maybe

__PCR #3: Squaring PCR mixture for FrTu target (third attempt)__
July 3, 2012 Lane 1: skip Lane 2: 100 bp ladder Lane 3: PCR sample a Lane 4: PCR sample b Lane 5: PCR sample c Lane 6: PCR sample d


 * Note that this sample actually did work, finally. I will continue on to next step soon - the purification step.

__PCR #3: Squaring PCR mixture for FrTu target (second attempt)__
July 2, 2012

[[image:ADO PCR 2.png]]
Lane 1: skip Lane 2: 100 bp ladder Lane 3: PCR sample a Lane 4: PCR sample b Lane 5: PCR sample c Lane 6: PCR sample d


 * note that this run looks almost similar to the previous run. I will make a new secondary PCR to run my next attempt.

__PCR #3: Squaring PCR mixture for FrTu target (first attempt)__
June 28, 2012

Lane 1: skip Lane 2: 100bp ladder Lane 3: PCR sample 1 Lane 4: PCR sample 2 Lane 5: PCR sample 3 Lane 6: PCR sample 4


 * note that there was an original 200ul of sample prepared then 50ul of it was then placed in 4 diff. tubes and were run as is. Also, the samples may have come out relatively dim because I may have not added enough dye to the samples. Samples 1 and 3 can be seen, but 2 and 4 are almost invisible.

__PCR #3: Second Attempt__
June 26, 2012

[[image:ADO pic 2.png]]
Lane 1: empty Lane 2: 100 bp ladder Lane 3: Primary PCR Lane 4: empty Lane 5: Secondary PCR

for the secondary PCR which had failed the first time it was run. Next step is the squaring procedure of PCR.
 * note that this run actually worked. Source of error could have been from using the incorrect forward and reverse primers

__PCR #3: First Attempt using Oligo mix for FrTu (Francisella Tularensis)__
June 22, 2012 Lane 1: empty Lane 2: 100 bp ladder Lane 3: Primary PCR Lane 4: empty Lane 5: empty Lane 6: Secondary PCR


 * note that the primary PCR ran ok, but the secondary failed. I will run this PCR again.

__PCR #2: pGFP__
//Aldo & Rishi PCR-pGFP//

Lane 1: Skip Lane 2: 5 ul 100 bp ladder Lane 3: 0.016 ng total pGFP plasmid in 25 ul total test tube Lane 4: 0.16 ng total pGFP plasmid in 25 ul total test tube Lane 5: 1.6 ng total pGFP plasmid in 25 ul total test tube Lane 6: 0 ng total pGFP plasmid in 25 ul total test tube Lane 7: 0.016 ng total pGFP plasmid in 25 ul total test tube Lane 8: 0.16 ng total pGFP plasmid in 25 ul total test tube Lane 9: 1.6 ng total pGFP plasmid in 25 ul total test tube

__PCR #1: pGBR22 plasmid__
Aldo & Rishi PCR-pGBR22 Lane 1: Skip Lane 2: 100bp DNA ladder Lane 3: 1 ul of 0.3 ng total pGBR22 plasmid Lane 4: 10 ul of 3.0 ng total pGBR22 plasmid Lane 5: 10 ul of 3.0 ng total pGBR22 plasmid in 25 ul total test tube

__Transformation Efficiency Results (Aldo, Rishi, Janice):__
Plate C: 1ng of Plasmid, 4000 colonies/ng

Plate B: 5ng of Plasmid, 2000 colonies/ng

Plate A: 25ng of Plasmid, appx. 1600 colonies/ng

__**Restriction Enzyme Digest Gel result (first wells are failure, dropped gel):**__
6/8/2012 Aldo & Alex Lane 1: skip Lane 2: 1 kb DNA ladder Aldo’s Lane 3: uncut pGBR22 Lane 4: EcoRI cut Lane 5: PvuII cut Lane 6: EcoRI + PvuII cut Alex’s Lane 7: uncut pGBR22 Lane 8: EcoRI cut Lane 9: PvuII cut Lane 10: EcoRI + PvuII cut

__**Nanodrop Results for pGBR22 plasmid, concentration = 426.3 ng/ul:**__
061212 - Aldo, which plasmid are these Nanodrop results for? -- DR. B Measurement 1: Concentration: 44805 ng/ul 260/280: 1.89 260/230: 2.47

Measurement 2: Concentration: 469.3 ng/ul 260/280: 1.90 260/230: 2.39

//__**Averages**__//: //Concentration: 458.9// //260/280: 1.89// //260/230: 2.43//

__**DNA Sequencing Results of First Run:**__
__**First Sample (ADO1):**__

NNNNNNNNNNNNNNGCGATTGGGCCCGACGTCGCATGCTCCCGGCCGCCATGGCCGCGGGATTTTAGTGATGGTGATGGTGA TGACCGAGCAAAGAGTGGCGTGCAATGGATATTTCACACTGCTCAACAAATGTGTAATCCTTGTTGTGACTGGTTACATCCA ATGGTGATGGTGATGACCGAGCAAAGAGTGGCGTGCAATGGATATTTCACACTGCTCAACAAATGTGTAATCCTTGTTGTGACTGGTTACATCCA ACCACCTCCTTCCAACTTCAGAGCCATAAAGTTGTTTCCTATCAGCATTCCATCTCGTGCAAAGAGACGCTCAGTGTTGGGT TCCCAGCCCTGTGTCTTCTTCTGCATAACAGGTCCATTGGGAGGAAAGTTCACACCAGAGATTTTGACATTGTAGATGAAAC AGTTGCCTTGGATGCTGGAATCATTGCTGACAGTACACACTGCACCATCTTCAAAGTTCATGATCCTCTCCCATGTATATCC CTCAGGGAATGACTGCTTTACATAATCAGGGATGTCTTCAGGGTACTTGGTGAATGGTATGCTTCCGTATTGAGACAGTGGT GATAAAATATCCCAAGCAAATGGCAGAGGTCCACCCTTGGTGACAGTGAGCTTTACCGTCTGCTCCCCCTCGTAAGGCTTTC CTTTTCCATCGCCTTCGACCTCAAAGTAGTGTCCATTGACCGTGCCTGACATATAAACCTTGTAGGTCATTTGTTTAGCGAT CACACTCATGATATTTCTCCTTCAATCAATCAAAATCACTAGTGCGGCCGCCTGCAGGTCGACCATATGGGAGAGCTCCCAA CGCGTTGGATGCATAGCTTGAGTATTCTATAGTGTCACCTAAATAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTG AAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGANC TAACTCANATTNATTGCGTTGCNCTCACTGCCCGCTTTCNGTNNNNAANCTGTCNTGNNGCTGCNNNNNANNNNNNNNNNNG GGNNNAGGNNNTNGCGNNTGGGNNCTNNNCNNNNNTCNNNNNNTGANTNNNNNNNNNNGNNCNNTNNGNTGNGNNNNNNNNN CNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNANNNNNNNNNNANNNNNNNNNNNCNNNAANNNNNTNNNNNN NNTNNNNNNNNNNNNNNNNNNNN

__**Second Sample (ADO2):**__

NNNNNNNNNNGANNATAGAATACTCAAGCTATGCATCCAACGCGTTGGGAGCTCTCCCATATGGTCGACCTGCAGGCGGCCG CACTAGTGATTTTGATTGATTGAAGGAGAAATATCATGAGTGTGATCGCTAAACAAATGACCTACAAGGTTTATATGTCAGG CACGGTCAATGGACACTACTTTGAGGTCGAAGGCGATGGAAAAGGAAAGCCTTACGAGGGGGAGCAGACGGTAAAGCTCACT GTCACCAAGGGTGGACCTCTGCCATTTGCTTGGGATATTTTATCACCACTGTCTCAATACGGAAGCATACCATTCACCAAGT ACCCTGAAGACATCCCTGATTATGTAAAGCAGTCATTCCCTGAGGGATATACATGGGAGAGGATCATGAACTTTGAAGATGG TGCAGTGTGTACTGTCAGCAATGATTCCAGCATCCAAGGCAACTGTTTCATCTACAATGTCAAAATCTCTGGTGTGAACTTT CCTCCCAATGGACCTGTTATGCAGAAGAAGACACAGGGCTGGGAACCCAACACTGAGCGTCTCTTTGCACGAGATGGAATGC TGATAGGAAACAACTTTATGGCTCTGAAGTTGGAAGGAGGTGGTTACTATTTGTGTGAATTCAAATCTACTTACAAGGCAAA GAAGCCTGTGAGGATGCCAGGGTATCACTATGTTGACCGCAAACTGGATGTAACCAGTCACAACAAGGATTACACATTTGTT GAGCAGTGTGAAATATCCATTGCACGCCACTCTTTGCTCGGTCATCACCATCACCATCACTAAAATCCCGCGGCCATGGCGG CCGGGAGCATGCGACGTCGGGCCCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTG ACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTANTAGCGANNANGN CCNCANCGATCNCCCNTCCCANCAGTTGCNCANNNGAATGNNNATGGNNNNNNCCNNNANNNNGCATNNCNNGGNGGGNGTG GNNNNNCGCNCAGCGNGACNCTANNNNNCNNNNCCNANNNCCNNNNNTNNNNNNNNTNNNNNNNNGCNNNNTNNNNNNNCCG TNANNNNAANNGGGNNNNNNNNNNNCNANTNNNNNTNNNNNNCNNNNNNNANNNNNNNGGNNNNNNNNNNNNNNNNNCNNNN NNNNNNANNNNNNGN