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Dilutions Protocols Draft
Dilutions Protocols Draft
Calculations
1.1
2
3
r3
2
(2503 106 )3 = 3.272 1011 m3
3
(1)
(2)
1.2
Mass of aptamer
MW NS1 Aptamer with biotin and 6T = 12,116 g/mol S15 was 34 BP and NS1 aptamer is 32 BP,
both form g-quadruplex tertiary structures.
S15 sequence: 5 GCACCGGGCAGGACGTCCGGGGTCCTCGGGGGGC 3
Mass in grams for S15: 10646.79 Da = 1.7679409179e-20 grams
Molar Weight S15: grams/mole = (1.7679409179e-20 grams) * (6.022140857(74)1023 mol1) =
10,646.8969 g/mol
1.3
1.4
(3)
Assume 2 uM, 10 uM, 20 uM final concentration of NS1 aptamer in solution (from Goda papers
aptamer concentrations)
2 uM = 2 106 M
10 uM = 10 106 M
20 uM = 20 106 M
30 uM = 30 106 M
20*106
20*106
20*106
30*106
1.5
1.6
1.7
1.8
Number of tests
1.9
1.10
1 mM MgCl2, 1X PBS, pH 7.5 (137 mM NaCl, 2.7 mM Potassium Chloride, 8 mM Sodium Phosphate dibasic, 2 mM Potassium Phosphate monobasic)
1 mM MgCl2 - 100 ml (1 M) : MW - 95.211 g/mol
1mL of solution - 100 buffers
137 mM NaCl : MW - 58.44 g/mol
.137 M = x g / L * 1/58.44 g/mol = 8 g/L
2.7 mM KCl : MW 74.5513 g/mol
.0027 M = x g / L * 1/74.5513 g/mol = .2013 g/L
8 mM Sodium Phosphate dibasic 250G : 141.96 g/mol
.0008 M = x g / L * 1/141.96 g/mol = .113568 g/L
2 mM Potassium Phosphate monobasic 500 g : 136.09 g/mol
.0002 M = x g / L * 1/136.09 g/mol = .027218 g/L
1.11
1.12
SB Solution: concentrations/dilutions
1.13
1.14
1.15
2
2.1
Protocols
Aptamer Reduction Protocol
1. xBriefly centrifuge the aptamer storage tube to ensure the dried aptamer pellet is at the bottom
of the tube.
2. Resuspend the aptamers pellet in TE buffer (10 mM Tris HCl, 0.1 mM EDTA, pH 7.5) or
non-DPEC treat-ed, nuclease free water to achieve 100uM concentration (using volumes indicated
in the Product Information Sheet that accompanies each order).
3. Incubate at room temperature for 30 minutes.
4. Briefly vortex and quickly spin the sample down ( 10,000g for 1 second).
4
2.2
1. Prior to use, dilute the reduced thiol-aptamer to its working concentration in Folding Buffer.
2. Heat the aptamer solution to 85 95 o C for 5 minutes.
3. Allow the solution to cool to room temperature ( 15 minutes).
2.3
Immobilize thiol-modified DNA aptamers in a Reaction Buffer overnight, followed by rinse with
water. Backfill the remaining gold surface with SB solution, followed by rinse with water.
2.4
Use varying concentration of NS1 in Measurement Buffer ie. go from 0-1400 nm over time and see
the signal difference.
2.5
To determine the binding efficacy of the DENV2 NS1 to its related aptamer, well be performing
something similar to an ELISA assay. This document will provide details on how this will be
performed.
Process (components, paragraph form): The process will work much the same as a standard
ELISA assay. The base of our assay will be a layer of gold plating. This is necessary because the
next component, the NS1 aptamer, is thiolated. Thiols bind to gold, thereby anchoring the aptamer
to the gold surface. The next component will be for the NS1, since it binding to the aptamer is
what we want to test. This NS1 well be using has another feature that will be important for this
assay: these particles have 6xHis tags attached to them. Once an adequate amount of time has
passed, the NS1 will have theoretically bound to the aptamer, we will add the antibody detector.
This antibody binds to His tags and will have a dye bound to the functional end. After washing,
what we expect to see is fluorescence (green light) from the bound dyes, which will show that the
NS1 is bound to the aptamer.
Process (components, layered from top to bottom w/ associated quantities needed*):
6x-His tag antibody (DyLight 488) NS1 protein, His-tagged NS1 aptamer, thiolated Gold surface
Process (general ELISA steps):
Details taken from ThermoFisher: https://www.thermofisher.com/us/en/home/life-science/proteinbiology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overviewelisa.html
The direct detection method uses a labeled primary antibody that reacts directly with the
antigen. Direct detection can be performed with antigen that is directly immobilized on the assay
plate or with the capture assay format. Direct detection is not widely used in ELISA but is quite
common for immunohistochemical staining of tissues and cells.