Began by precipitating 40 hr samples by non-PEG method. 50 minutes, 20.0 xG. A visible precipitate formed in the 6 and 12 hb. The supernatant was removed and was prepared to go to Sullivan lab for plate reading. The Daunorubicin/Agrose gel mixture was tested on the PCR to get comparable readings. All supernatant samples were numbered as below.
At the Sullivan lab the samples were prepared in the plate with A3-H3 and A4 being the unknowns and A5-F5 the Dauno/gel mixture and A6-F6 the Dauno/gel/PEG mixture. G5 and G6 were set up with water for the bottom end. The Standards were used to measure each of the unknowns. Every amount was diluted from 20 uL to 100 uL for proper reading in the plate reader. Results will be inserted and analyzed via Excel.
A3 - 1-5 -----6 hb Trial 3
B2 – 6-8 -----12 hb Trial 3
C3 – 9-14 ----- BL Trial 3
D4 – 15-21 ------BL PEG trial 3 (Might be throw out worthy)
E5 – 22-24, 30-33 -----BL Trial 2
F5 – 50-54 ----- BL PEG Trial 2
G5 – 28-29, (44-47)(PEG) -----18 hb Trial 2
H5- 25-27, (48-49)(PEG) ----- F18 hb Trial 2
A4 – Sample 48 = Structure in 300 uL PBS
34-35, 37-38,40-43 ----- BL 1st trial
36,39 ------Hinge 1st trial
Ran gel of 8 Benet Linkage to purify the DNA
Cut the gel and mixed with Daunorubicin (2mM)
Made a 2mM solution of DR from other 10 mM, 117µL stock solution
Also mixed other Benet Linkages, 18 Helix bundles, and flat 18 helix bundles
Made 184.4 µL of 10mM DR stock solution
Performed half of Trial with PEG to increase visibility of precipitate and other under same procedure as before
Suspended structures in 50µL of PBS.
Began centrifuging – ran for 50 minutes at 20˚C at 20 g’s. The solutions that were used were OBL18 and CBL22 which were incubated at around 39 hours.
And then was run through the PCR “trial-1-39hrs” folder in Desktop->Biomod->Results. The results show the concentration of DR in the supernatant compared with a only DR solution. The results show that the supernatant of the DNA origami and DR mixture to have lower concentrations of DR compared with only DR.
Began to centrifuge the incubated solutions containing DNA origami and DR (45-46 hours of incubation) at 20 g’s for 50 minutes with the temperature at 20˚C We saw a color of reddish pink mixture after centrifuging. We separated 20µL of the supernatant out of the origami and DR mixture. OBL 18 and CBL 22 were used. The precipitate will be used to receive the absorbance numbers. In small wells 20 µL of each supernatant solution is placed separately along with 20µL of DR. Take absorbance readings put the supernatant in another tube. In the other tube with the DNA origami add 200 µL of PBS to buffer the solution and store for future use.
The decision was made to check some of the samples at an earlier time at 1:00 PM today one hinged; one CBL and one OBL were centrifuged. There was definitely a visual change in the color becoming a pink hue. Centrifuged for 10 minutes at 10 k RPM was insufficient for precipitation it was run at 20,000 RPM for 30 minutes, some had a visual precipitation. Ran 20 trials through PCR (20 μL each sample) with the control subastance 20 μL of Danuorubicin 2 mM. Results are under the folder BioMOD on PCR computer.
The 24 hours samples were centrifuged for 45 minutes at 20 °C at 20 g’s. The supernatant was removed and run through the PCR again 20 μL.
A1 – Hinged
B1 – OBL
C1 – CBL v D1 – Dauno 2mM
Closed benet linkages = CLB
Open benet linkages = OBL
Hinges = Hin
Today we are trying to purify the DNA using the gel electrophoresis method. We followed the lab protocol. In the first gel we have:
CLB20 CLB 20 CLB22 CLB22 OBL18 OBL18 OBL18 OBL18 HIN14 HIN16
In the second gel we have:
CLB20 CLB22 CLB22 CLB22 OBL18 OBL18 OBL18 OBL18 HIN18 HIN20 HIN22
Incubation started at 7:16
48 hours: 1CBL 1 OBL
36-40 hours: 1CBL 1 OBL
24 hours: 2CBL 2 OBL
We made 585 μ of 2mM Daunorubicin out of the 10 mM supply leaving 117 μL left of the 10 mM. I’ve then mixed together 20 μL of Daunorubicin with approximately 20 μL of the nanostructures, which are approximately 5.0 nM. They are being incubated at 4°C for various times which are listed above.
The gels were each cut individually and labeled, then centrifuged for 10 minutes at 4° C at 10000 RPMS. The samples in gel # 2 are not reliable and will not be used at this time.