Biomod/2013/LMU/protocols

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Contents

Imaging and Analysis of our Samples

TEM

Grid Preperation

Grid Preperation

  • plasma cleaning (to ensure grid surface is hydrophilic)
    • 60s, 240V

Sample preperation

  • pipette 2µL of sample on grid surface and wait at least 60s (depending on the sample concentration)
  • dab excess liquid onto filter paper
  • make sure the grid is completely dry!
  • allow grid to air-dry for 30min

Uranyl Acetate Staining (for TEM)

Uranyl acetate

  • 1% Uranyl acetate in H2O
    • tumble in fridge overnight (optional)
  • filter solution using 0.22 μm filter
  • store in fridge protected from light

Grid Preperation

  • plasma cleaning (to ensure grid surface is hydrophilic)
    • 60s, 240V

Sample Staining

  • for each sample, place 2 droplets of uranyl acetate (7µL each) on parafilm
  • pipette 2µL of sample on grid surface and wait at least 60s (depending on the sample concentration)
  • dab excess liquid onto filter paper
  • briefly submerge grid into the first droplet and dab excess liquid immediately onto filter paper
  • submerge grid 15s into the second droplet and dab excess liquid onto filter paper
  • make sure the grid is completely dry!
  • allow grid to air-dry for 30min

Gel Analysis

PAGE Gel

Content Volume
Page Gel 30% 4ml
5x TBE 1,2ml
H2O 6,7ml
TEMED 8µl
APS 88µl
Total 12ml


  • prepare gel according the volumes in the table above
  • Pour gel into casting tray (no bubbles!), and insert comb
  • wait about 20 min
  • Fill in TBE-Buffer
  • Than: Run the gel by 120 V and 30 min!


Agarose Gel

  • Measure 1.05 g for 0.7% gel agarose in 250mL flask.
  • Add 150 mL 0.5X TBE up.
  • Microwave for ~3 minute (600W) to disolve the powder
  • Gently swirl in an ice water bath until it is not hot for your hand
  • If running a MgCl2 gel, gentally add 1.5 mL of 1M MgCl2 (final concentration = 10 mM).
  • Pour gel into casting tray, and insert comb with the thick teeth pointing down.
  • cast the gel and cool the whole setup with ice water down to about 4°C
  • Prepare the sample by adding 6× loading buffer containing Orange G (school bus orange, 50 bp) and sucrose
  • Set to 60 V, run for 1.5 hours

Absorption Spectrum

Nanodrop

Spectrophotometric Measurement of Nucleic Acid Concentration

Spectrophotometric conversions for calculating the concentration of nucleic acids from their absorbance at 260 nm (A260)

Table 1. Spectrophotometric conversions

1 A260 unit Concentration (μg/ml)
dsDNA 50
ssDNA 33
RNA 50
Oligonucleotides 20–30

This relationship is only valid for measurements made at neutral pH, and is based on a standard 1 cm path length.

An example of the calculation involved in RNA quantification is shown below:
Volume of RNA sample = 100 μl
Dilution = 10 μl of RNA sample + 490 μl distilled water (1/50 dilution).
Measure absorbance of diluted sample in a quartz cuvette.
A260 = 0.23
Concentration of RNA sample = Spectrophotometric conversion x A260 x dilution factor
= 40 x 0.23 x 50
= 460 μg/ml
Total yield = concentration x volume of sample in milliliters
= 460 μg/ml x 0.1 ml
= 46 μg

Spectrophotometric measurements must be taken using a quartz cuvette. If you use more than one cuvette to measure multiple samples, they must be matched. To ensure readings fall within the linear range, values should lie between 0.1 and 1.0. Dilute samples in a low-salt buffer with neutral pH (e.g., 10 mM Tris·Cl, pH 7.0) when making spectrophotometric measurements for determining nucleic acid concentration. Spectrophotometric quantification of DNA is accurate only when the sample is not contaminated with RNA, and vice versa. Phenol has an absorbance maximum of 270–275 nm, which is close to that of DNA and RNA. Phenol contamination mimics both higher yields and higher purity because of an upward shift in the A260 value.

Assembly of DNA-Origami

see Origami-Design

Functionalizing of ND's

Direct coupling

  • Nanodiamonds in ultrasonicator (the ultrasonicator helps to break the clusters of nanodiamonds, although the reunification is expected to set in after a few hours)
  • solution of acid treated nanodiamonds
  • mixing the solution for 5 min
  • add EDCi (EDCi reacts with carboxyl groups and forms an active intermediate. The EDCi belongs to the family of EDC coupling chemistry, however has an even more aggressive active intermediate, which ultimately reacts with the attacking amine)
  • mix for 15min
  • add 60 μl of amine-DNA.

Coupling of DNA to magnetic nanoparticles

Conjugation of neutravidin to ND

Preperation:

  • use DNA low-bind tubes
  • dilute neutravidin in coupling buffer (ocean nanotech) to a final concentration of 2.8 mg/ml
  • freshly prepare a undisolved mixture of EDAC cloride/ Sulfo-NHS (in a ratio of 2:1)

Conjugation:

  • mix 16µl of ND with 16µl of activation buffer (ocean nanotech)
  • disolve the EDAC cloride/Sulfo-NHS mixture with the activation buffer to 2/1 mg/ml and add 8µl to the ND solution
  • mix well for 15min
  • add 40µl of coupling buffer, mix quickly and directly afterwards add 40µl of the prepared neutravidin solution
  • mix for 2hrs

Purification:

  • Fill the solution up to 500µl with TBE 0.5x/Tween20 0.05% buffer and centrifuge at 14.6k for 10min
  • directly afterwards carefully put the epi over a permanent and pipet away the surfactant
  • fill up to 500µl with TBE 0.5x/Tween20 0.05% buffer and centrifuge at 14.6k for 10min
  • repeat for another two times

Conjugation of biotin-DNA to neutravidin modified ND

Coupling:

  • Mix 80µl of TBE 0.5x/Tween20 0.05% with 10µl of biotin-modified DNA oligos (15T / concentration: 100µM)
  • add 10µl of the ND
  • mix well for at least 30min

Purification:

  • Fill the solution up to 500µl with TBE 0.5x/Tween20 0.05% buffer and centrifuge at 14.6k for 10min
  • directly afterwards carefully put the epi over a permanent and pipet away the surfactant
  • fill up to 500µl with TBE 0.5x/Tween20 0.05% buffer and centrifuge at 14.6k for 10min
  • repeat for another two times

BSA functionalization and Coating

According to the protocol for coating:

  • Mix 200µl of BSA (100µM) with 200µl of UREA (10M) for 5 min
  • add 88µl of TCEP(100mM) and wait for 30min
  • add biotin-PEG with 22,2mg/1ml, 17ml of 0,5xTBE and mix for 1,5h
  • add NAM with 2,2mg/1ml and wait 1,5h
  • finally mix the solution with 400µl of nanodiamonds and wait for 6h
  • use Amicon Filter for purification

Buffers and Media

5xTBE Buffer

per 1 Liter of 5xTBE stock solution (pH 8.2)

  • 54.0g Tris-base
  • 27.5 g Boric Acid
  • 20mL 0.5M EDTA pH 8.0 (or 2.92 g EDTA sodium salt)
  • Add ddH2O to 1L
  • optional: 22.36 g MgCl2·6H2O for a final concentration of 11mM in 10L

Filter Methods

Gel Purification

After running a gel (cf. Agarose Gel), the following procedure describes a method to separate material in the gel that has a distinct status of modification:

  • Cut out the distinct area of gel, where the bands that are useful, to be expected
  • put it in a bath of 0.5xTBE with staining material SYBR gold
  • place the bath with the gel on a shaker and run the shaker on slow speed for about 30min
  • image the gel under UV light
  • cut out the gel fragment according the sample band
  • squeeze the material carefully out of the gel using cover glass and pipette away the liquid solution on the glass edge
  • redo the last two steps until the gel is used up

Amicon Filter

This procedure is for purification of solutions using the Amicon filters. Usually we use the filters with a 100K to get rid of free material after reactions(depending on the size of the free material):

  • Fill up the Amicon filter with the solution
  • collect the sample by centrifugation at 3,000rpm, 4°C for 3min (depending on the concentration)
  • after centrifugation, remove waste material
  • redo the process until all solution is used
  • pipette solution in the filter and put it in a new tube
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