Dec 19, 2023
Diamond XChem Seeding Protocol
- 1Diamond Light Source
- ASAP Discovery
Protocol Citation: Peter Marples, Charlie Tomlinson, Daren Fearon 2023. Diamond XChem Seeding Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx3nwog8j/v1
Manuscript citation:
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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
We use this protocol and it's working
Created: October 06, 2023
Last Modified: December 19, 2023
Protocol Integer ID: 88908
Keywords: Seeding, Crystallisation, XChem
Funders Acknowledgements:
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
Grant ID: U19AI171399
Disclaimer
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Abstract
The use of seeding in protein crystallisation is a highly effective method for increasing the quantity of usable crystals from a single plate and can be crucial for obtaining crystals in a highly reproducible manner. Even crystallisation experiments which did not previously require seeding can benefit from the use of seeds when transferring protocols between labs.
Image Attribution
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Guidelines
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Materials
Formulatrix Protein Crystallization Imager
SPT mosquito
P10 pipette
SwissCI 3 lens plate
Seed beads
Crystalline material
Vortex
Safety warnings
Follow safety guidlines in regards to the chemicals present in the protein buffer and crystallisation condition.
Ethics statement
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Before start
Prepare crystalline material needed to produce crystals.
Equiptment needed
Equiptment needed
Formulatrix Protein Crystallization Imager
SPT mosquito
P10 pipette
SwissCI 3 lens plate [UVXPO-3LENS3W96T-PS3W96T-UVP]
Seed beads [Hampton HR4-782]
Crystalline material
Vortex
0.5 mL Eppendorf tubes [Eppendorf Catalog No. 0030121023]
Seeding experiment
Seeding experiment
17m 40s
17m 40s
Produce crystals to use for seeding. Micro crystalline protein material can be used to produce a seed stock as well.
Set up the serial dilution for step 10 using the reservoir solution used to obtain the starting crystalline material On ice . Make up an appropriate volume; most seeding uses 20-50 nL of seed stock, meaning a SwissCI 3 lens 96 well plate could require ~14.5 µL of seed stock per plate.
5m
Add seed beads to the undiluted tube in the serial dilution (Check manufacture instructions).
For Hampton glass beads, use 2 beads in a 0.5 mL Eppendorf tube
1m
Under a microscope, open the well containing the crystal material being used and crush the crystals. From this step onwards proceed rapidly as the seed stocks are metastable and need to be frozen as soon as possible.
1m
Pipette2 µL from the reservoir well into the drop well
20s
Mix the solution by aspiration a minimun of three times
10s
Transfer to the first tube set up for the serial dilution that will remain undiluted and is marked "undiluted seed stock"
10s
Repeat steps 5-7 until there is no more crystal in the drop well. To ensure all available crystal material is recovered, a minimum of around 30 µL of reservoir solution should be used in this process
2m
Vortex the undiluted tube containing the seed beads – 30 seconds on vortex then 30 seconds on ice, repeating three times. Take 2 µL of the seed stock and check under microscope whether crystals have been crushed.
3m
Carry out the set up serial dilution using the undiluted seed stock and freeze stocks at -80 °C
5m
Interpreting results
Interpreting results
Based on what your project needs, decide which condition made from the serial dilution makes the best results with good reproducability. If needed, further optimise around the dilution e.g. if 1/100 works best, try using 1/20, 1/50 and 1/200; repeat until crystals are acceptable for your project and its timeframe.