The iron salts were added to an Erlenmeyer flask with water to create a solution. This was stirred at 800 RPM.
            1:30 – start of the addition of NH₄OH.
            2:15 – end of addition of NH₄OH.
     Heating at 90°C was started.
            3:00. Heating and agitation were stopped and the sample was washed twice with water, precipitating the nanoparticles with an external magnetic field. 50 ml of sodium citrate 10mM were added.
     The sample was ultra-sonicated for 2:30 min at 65% amp with a probe.
     The solution was washed with water to remove any excess of sodium citrate and the size was measured by Dynamic Light Scattering (DLS). The biggest peak was found between 69-70nm. The sample was dried under vacuum overnight.

      03/07/2014

     Once dried, the sample was ground with a mortar and then coated with TEOS.
          Coating – 50 ml of absolute EtOH and 10 ml water were mixed with 500 mg of Fe₃O₄ nanoparticles and were ultra-sonicated at 70% amplitude using a sonic probe.
              100 μl of TEOS were dissolved in 10 ml of absolute ethanol. After, this solution was added dropwise to the nanoparticles, stirring at 750 RPM.
     The addition of TEOS lasted 20 min and the nanoparticles were left stirring overnight.

     04/07/2014

     The same procedure was followed, changing the amount of NH₄OH for 10 ml 5M and adding this amount dropwise over 15 min. Afterwards, the addition of sodium citrate was repeated. (MB2)
     15 ml of the ferro-fluid were diluted in 30 ml of ethanol for coating with 150 and 100 μl of TEOS each. (SBS_1 and SB2_2). Each volume of TEOS was diluted in 10 mL EtOH and added dropwise under intense magnetic stirring.

     07/07/2014

      The same procedure was followed a third time for the coating with TEOS. This time 75 μl were used and left 3 hours in constant stirring (SB2_3). Functionalization with APTS.
     15 ml of the magnetic fluid were washed with isopropanol (20 ml) and diluted in toluene with 100 μl APTS. (NB2)
     The average refractive index was calculated as follows:

Fe₃O₄, n = 2.42
SiO₂, n = 1.46
2.42 + 1.46 = 3.15

     08/07/2014

     Samples SB2_1, SB2_2 and SB2_3 were washed with water three times and were sonicated with a sonic probe for 1 minute at 60% amplitude.
     Once again, it was observed how the solid was easily dispersed in water, forming a fake solution (colloid). Spinels were observed in the presence of a magnetic field with samples NB2 and SB2_(1-3).

     09/07/2014

     The nanoparticles were characterized by DLS. For MB2, it was observed a peak at 39 nm, once coated with TEOS, the peak was moved toward 60 and 80 nm.
     For different amounts of TEOS, same results were achieved. (50, 75 and 100 μl).

     10/07/2014

     The samples SB2_(1-3) were evaporated until the amount of water was minimum. The last residual water was removed with absolute EtOH for the correct functionalization with APTS.
     15 ml of absolute ethanol were added to the sample SB2 in intense agitation. Finally, 75 μl of APTS were added and left in constant stirring overnight.

     11/07/2014

     The samples were once again washed with water, these samples easily precipitated in water, contrary to previous samples. Code names for these samples were NB2_(1-3)
     To help the precipitation, a little amount of acetone was added, this was also done for sample SB2’s aliquots.
     The samples were magnetically decanted and dried under vacuum for 4 hours.

     15/07/14

     Samples were taken of pure magnetite, silanized magnetite (NB2) and magnetite coated with silica and silane (SB2_1 and SB2_3) for its characterization in IR for solids.
     Comparing the spectra given by the IR of the pure magnetite and silanized magnetite (SB2 and NB2) we were able to distinguish a peak at 990.2 cm^-1 corresponding to a Si-O bond, confirming the correct silanization of the magnetite, although the amino group couldn’t be identified.
     Similar results were achieved for samples SB2_1 and SB2_3, however, this peak was not decisive to know if the sample was correctly functionalized giving the fact that the samples are coated with silica.

     29/05/2014

     For the first day of wetlab, we prepared the solutions we would use throughout our project.
     

• TE Buffer Recipe:
1. 10 mM Tris, pH 8 (with HCl)
2. 1 mM EDTA
• To make the CTAB/NaCl solution:
1. 4.104 g of NaCl
2. Slowly add 10.004 g CTAB
3. Add 60 ml of water
4. Heat at 74 °C

     After, we prepared a solution of EDTA 0.5 M by adding 3.65 grams in 25 mL of water.
     The Tris 1M solution took 3.02 grams in 25 mL water. These would be further diluted later on.
     A mistake was made within the calculations, a redo would have to be performed. The correct amount of EDTA and Tris were weighed and left for later.
     The day lasted longer than anticipated, the task list involved:
     

• Hydration of the EDTA and Tris
• Review Tris’ pH with potentiometer
• Mix TE buffer
• Sterilize solutions

 
 
 
 

     30/05/2014

     After the Tris hydration, a pH of 6.55 was obtained. What was to follow was to achieve a pH of 8 by neutralizing with NaOH.

     Sudden realization came when the pHmeter was reviewed; it wasn’t working, so the pH was, in fact, much higher than the one figured on display. The next step was to acidify by dropwise adding of HCl.

     In order to dissolve the EDTA in water, it was needed to raise the pH up to 8 (it was at 3.83)

     The first of many sterilizations took place (1 box of blue tips, 1 box of yellow tips, TE Buffer and EDTA
     Both solutions remained at room temperature after sterilizing.

 
 
 
 
 
 

     03/06/2014

     DNA extraction of E. Coli began:

1. The bacteria was grown in an LB medium, obtaining two distinct batches.
2. The bacteria from the 30^th was used.

     The vacuum chamber was cleaned with EtOH and kept under UV light for 20 minutes. To each tube, 567 µl of TE and 30 µl of proteinase K 20 mg/ml were added. This was incubated in oven at 37 °C for an hour, then placed in the refrigerator

     09/06/2014

     As proteinase K supply was running low, a distinct method had to be pursued.

1. The bacteria-rich broth was centrifuged: @5000 rpm, 10 minutes, 4 ºC
2. 5 ml of TE buffer were added, then centrifuged: @5000 rpm, 5 min, 4 °C
3. 5 ml phenol/chloroform/isoamylic solution were added after, the formation of organic phases took place, the two lesser dense were clear, the bottom one tinted a light pink.
4. Vortex spun for 3 minutes.

     The to-do list for the following day was as follows:

• Centrifuge at 6000 rpm, 20 min, 4 ºC
• Decant the supernatant
• Wash well with 0.5 ml of TE
• Transfer into Eppendorf tubes

 
 
 
 

     10/06/2014

     Part of the team centrifuged a day before. Re-centrifugation lasted for 10 minutes. The supernatant was cleared off. The Epperndorfs were left to dry out for about half an hour. After adding 0.5 ml of TE and two volumes of absolute EtOH, the DNA strings were not visible.
     We centrifuged at 1300 rpm, then a volume of 50 µl TE was added to ‘A’ labelled tubes and 200 µl to ‘B’ labelled tubes. We added DNA extracted with the first protocol to the ‘A’ labelled tubes and these were re-labelled ADN-1, labels of C-1 and C-2 were conscripted.

1. Another centrifugation: 6000 rpm, 10 min, 4 ºC
2. Removed supernatant
3. Dried out
4. 5 ml TE added, moved to Eppendorf tubes and two volumes of absolute EtOH
5. Re-centrifuged at 13,000 rpm
6. Left to dry
7. Poured 400 µl TE in tube C-1
8. Poured C-1 into C-2

     Electrophoresis was run on DNA to check in indeed DNA was present.

• The elaboration of the gel for electrophoresis 1x with 1% agarose:
• 49 ml of water
• 1 ml TAE (50x)
• 0.5 gr agarose
1. Mixed in an Erlenmeyer flask
2. Heated until no crystals were seen
3. Poured out onto the electrophoresis plaque, left it to turn into gel.
• The electrophoresis buffer, 250 ml:
1. 5 ml TAE (1x)
2. 245 ml of water

     11/06/2014

     Ran electrophoresis over 80 V for approximately an hour and a half.

     07/08/2014

     The protein finally arrived. WetLab time was on the go. We re-suspended the protein and prepared EDTA and Tris/acetate.

• To prepare TAE 2M
1. 24.2 g Tris base
2. 75 mL water
3. 5.7 mL conc. acetic acid.
4. 10 mL of EDTA

     Magnetite was dispersed in anhydric toluene followed by the addition of the resuspended protein with some triethylamine and finally glutaraldehyde was added as a coupling agent

     15/08/2014

     Alpha samples were divided into αG (G for glutaraldehyde) and αSG (without G), β samples were divided in the same way as α. Both G samples (α & β) were washed 3 times with PBS and some glutaraldehyde. The SG samples were both washed 3 times with pure PBS. UV spectra were taken for characterization.

     19/08/2014

     Proceedings to prepare TAE for electrophoresis using TRIS, acetic acid and EDTA were performed. Agarose gel was prepared with distilled water and agarose, a loading buffer was prepared with glycerol at 10%. The electrophoresis revealing did not quite displayed desired results, a new run had to be made.

     28/08/2014

     DNA extraction was performed anew:

1. E. coli was cultivated in LB broth for 24 hours at 37 °C, with constant stirring.
2. Pouring into Falcon tubes
• Tube 1. Miniprep procedure for extraction
• Tube 2. E. coli in 30 ml of TE, 20 ml of isopropylic alcohol and NaOH 0.1 M were added

     01/09/2014

     Transformation and preparation of DH5α was done.
     CaCl₂ was prepared to make competent cells. For a 200 ml solution, 54 grams of CaCl₂ were added. Also, chloramphenicol was needed, 34 micrograms for each 250 microliters.
     500 ml of LB broth was prepared with:

1. 5 gr Tryptone
2. 2.5 gr Yeast extract
3. 0.5 gr. NaCl

     03/09/2014

     The protein was washed 3 times in PBS and sonicated for 10 minutes. The protein was then suspended in PBS and some magnetite was added. The samples were separated in four groups, those containing DNA and those without, and within them ones containing glutaraldehyde and the rest without.

     04/09/2014

     Plasmid resuspension in 100 microliters TE or CaCl₂

     05/09/2014

     

• Transformation of pGLO/pSB1C3:

     E. coli was cultivated in 30ml of LB broth, then stirred and transferred into falcon tubes. Passed into the centrifuge and washed with cold CaCl₂, resuspended and recentrifuged.
     

• pGLO

     E.coli was transferred into vials and resuspended pGLO was added.
     

• pSB1C3:

     Some more E.coli was transferred into vials and resuspended pSB1C3 was added. All the tubes were placed in an ice tray and then left at room temperature.
     These were incubated at 37Cº for 30 minutes and some samples were taken from each tube to cultivate on Petri dishes.

     09/09/2014

     

• DNA Purification:

     In the presence of ethanol it precipitates, it then was centrifuged and the supernatant disposed of. Resuspension on PBS followed and then passed onto an Eppendorf tube and finally adding some RNAse.

     17/09/2014

     

• Protein+DNA:

     Different dilutions of DNA were separated labeling samples as β and γ. These, as well, were divided with and without glutaraldehyde, and subsequently with and without DNA. We did some UV characterization for all the samples. All data was saved.

     21/09/2014

     +pGLO was cultivated in LB.

     24/09/2014

     1ml of cultivated LB was dropped in Eppendorf tubes along with +pSBC13

     25/09/2014

     Some washes with water of the β and γ samples, some PBS was put into the mixture as well.
     Original samples stay resuspended in water, and new samples are kept on the fridge. The samples for electrophoresis and for UV characterization were prepared.

     26/09/2014

     

• Preparation of mediums LB/CP/iPGT for a pSB1C3 promoter:

     First, the transformation was made. For this, E.Coli was cultured in LB broth, stirred at 37Cº, transferred into a Falcon tube for centrifuging at 1500rpm 4°C during 10 minutes, and resuspended in CaCl₂. Recentrifuged again and repeat. The suspension was left on ice for half an hour. Another centrifuging, and another resuspension, some sample was taken and passed into an Eppendorf tube containing +pGLO. Ice resting for another 30 minutes. Then, incubation in a stove at 37Cº.
     

• DNA Purification - MiniPrep:

     The DNA in Eppendorf tubes was centrifuged, forcing it to precipitate, then the supernatant was removed and EtOH was added in order to centrifuge again. The tube was left to dry for a few hours. The tube with the DNA was added to a previously prepared solution of PBS and RNAse. Placed then into the thermoblock for about 15 minutes to finish incubation.
     Finally, the tubes were centrifuged one last time and the sample separated in two, having two different concentrations.

     28/09/2014

     Some of the σ samples were prepared, one with glutaraldehyde and DNA-protein, another with glutaraldehyde and DNA; the third, with DNA-protein and the last one with DNA only.
     The ε samples were prepared likewise its σ counterparts. A and B samples were prepared with some nanoparticles.

     30/09/2014

     The A10, E10, C10, B10 samples were ran in an agarose gel at 1% and prepared to an electrophoresis run.

     Performed tests

     

• Electroporation by using the system on BBa_K737051 as control against psBC13 alone
• Electrophoresis using DNA as λ marker with an integrated colorant to the system
• Sonication of the complex with a λ marker and electrophoresis evaluation