Jan 26, 2016
Cesium Chloride Gradients
- Matthew Sullivan1
- 1Matthew Sullivan Lab
- VERVE Net
- Sullivan Lab
Protocol Citation: Matthew Sullivan 2016. Cesium Chloride Gradients. protocols.io https://dx.doi.org/10.17504/protocols.io.c7dzi5
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: June 24, 2015
Last Modified: March 15, 2018
Protocol Integer ID: 965
Abstract
The usefulness of cesium chloride (CsCl) step gradients and continuous gradients for the separation of viruses is based on the differing buoyant densities of viruses, bacteria, and extracellular debris. This protocol provides a method for Cesium Chloride and DNA Extraction for Viruses (See guidelines for DNA Extraction).
Guidelines
This protocol is part of a larger collection of Cesium-Chloride related protocols. This is number (1) of (4):
Needed:
- Cesium chloride
- Seawater that has been 0.02µm or 0.2µm filtered and autoclaved
- Balance
- Sterile pipet tip
- Gradient fractionator
- Centrifuge (SW40 or SW28 Beckman) @ 24,000rpm
- Sterile 0.5ml tubes
- Sterile 1.5ml tubes
- Rack
- Ring stand
- Sterile 20 guage needle
- 4-place balance
- SYBR Gold Nucleic Acid Gel Stain
- Fluorescent Scope
- Wizard DNA Purification Resin
- Wizard Minicolumn
- Syringe
- Plunger
- 1.5ml centrifuge tube
- 80% isopropanol
- TE buffer
DNA extraction
Wizard DNA Purification Resin (Promega #A7181)
Wizard Mini Columns (Promega #A7211)
Phage Buffer to dilute if necessary (150mM NaCl, 40mM Tris-Cl, pH7.4, 10mM MgSO4 in nuclease-free water; fliter sterilized)
Mix 1ml DNA Purification Resin with 0.5 ml CsCl sample (can use up to 1ml sample but more than that will significantly decrease yield of DNA). Attach minicolumn to bottom of 3ml or 5ml sterile syringe that has had plunger removed. Add resin with sample to the syringe and push through the solution (can save flow-thru just in case you think you overloaded the resin). Remove minicolumn from the syringe and pull out plunger. Reattach minicolumn to the syringe and 2ml of 80% isopropanol to the syringe. Using the plunger push through the isopropanol to wash the resin. Remove minicolumn from syringe and place in a sterile 1.5ml centrifuge tube. Centrifuge 10,000 g for 2min to remove any residual liquid. Place minicolumn in new sterile 1.5mil centrifuge tube. Add 100μl 80oC TE buffer to top of minicolumn. Place tube lid over top of column and vortex for 10 seconds. Wait another 30 seconds and then immediately centrifuge at 10,000 g for 30 sec to elute DNA. Can repeat this a second time using 50μl warm TE (do not pool the 2 elutions until you quantify so as not to dilute the sample). Usually can recover an addition 10-20% of DNA with the second elution.
Note: if you have more than 1ml of CsCl sample, you can use more Wizard columns, or you can concentrate prior to DNA extraction using Amicon Ultra Concentrators (100kDa MWCO). Try to use the size that fits most of your sample in one or two spins; spin at 1000g for 5 min at 10°C and check volume. If you need to add more volume to the retenate, use the flow through to do this.
Cesium Chloride gradients
Cesium Chloride gradients
Prepare cesium chloride densities of p1.2, p1.4, p1.5 and p1.65 in seawater (sw) that has been 0.02μm (or 0.2μm) filtered and autoclaved.
Place tube of prepared cesium on balance
Tare balance to 0
Remove 1ml with sterile pipet tip.
| p1.2 | 11.19g per 50ml seawater | |
| p1.4 | 26.94g per 50ml seawater | |
| p1.5 | 33.74g per 50ml seawater | |
| p1.65 | 43.78g per 50ml seawater |
1 mL
Note
Difference in weight should be equal to the density of the solution (eg, 1ml of p1.2 should weigh 1.2ml). Adjust as needed.
Layer CsCl from the bottom using a gradient fractionator if you have one or by hand:
| SW40 rotor: | 2ml p1.65 | |
| 3ml p1.5 | ||
| 3ml p1.4 | ||
| 1ml p1.2 |
| SW28 rotor: | 5ml p1.65 | |
| 8ml p1.5 | ||
| 9ml p1.4 | ||
| 3ml p1.65 |
Note
The p1.2 layer is to equilibrate sample before it hits the p1.4 cesium layer
Note
Use thin-walled SW40 or SW28 tubes!
Note
This was done with a fraction collector (Labconco) but you can carefully layer these solutions with Pasteur pipets.
Carefully layer on sample (~5ml can be layered with SW40 ~15ml with SW28).
Note
Use thin-walled SW40 or SW28 tubes!
~5ml can be layered with SW40, ~15ml with SW28)
Note
This was done with a fraction collector (Labconco) but you can carefully layer these solutions with Pasteur pipets.
Weigh and balance tubes.
Centrifuge using SW40 or SW28 Beckman rotor @ 24,000rpm for 4hr at 4°C
04:00:00
Note
With a SW-48 swinging-bucket rotor (Beckman), setting 9 was used for deceleration. This slower deceleration should reduce mixing in your gradient.
Have sterile 0.5ml tubes (SW40) or 1.5ml tubes (SW28) labeled 1-24
Open tubes in a rack.
Place tube on ring stand
Puncture about 2mm from the bottom side of the tube using a sterile 20 guage needle, bevel up.
Note
To make things easier, can pull off top sample layer with pipette as it will not contain any viruses after centrifugation; but drops will slow down as you reach the end of the collection.
Collect droplets to fill each tube in order.
Note
Do not need to collect bottom layer as virus will not be in that layer.
Place tube with sample on 4-place balance in holder.
Note
Make one by cutting off top 1" of 15cc tube with cap on.
Tare to 0.
With sterile pipet tip, remove 100µl.
100 µL
Take measurement.
Return sample to tube.
Remove another 100µl, take measurement, return sample to tube.
100 µL
Remove yet another 100µl, take measurement, and return sample to tube.
100 µL
After all tubes have been measured, take average of the 3 values for each tube.
Multiply by 10.
Note
This will equal the density of the CsCl in that tube. For viruses, collect the p1.4-1.5 samples.
Pool and extract DNA or can confirm presence of virus first by staining 10µl with SYBR gold
10 µL
Count under fluorescent scope.
Pool all samples with virus.