W-3 WATER STORAGE

Disclaimer

This work is supported by the US Army Medical Research and Development Command under Contract No.W81XWH-21-C-0001, W81XWH-22-C-0093 and HT9425-23-C-0059. The views, opinions and/or findings contained in this report are those of the author(s) and should not be construed as an official Department of the Army or Navy position, policy or decision unless so designated by other documentation.

Abstract

This protocol describes leech storage.

Steps

STORAGE PROCEDURE FOR UNTREATED SAMPLE

Note

NOTES : Collected water samples need to be kept on a cold chain all the time to prevent RNA degradation. The following procedure will apply only where -80°C storage is feasible.If -80°C storage is not possible, temporarily store the soil samples in a -20°C freezer and follow water sample processing SOP (REDI-NET Water Processing SOP W-2) as soon as possible for total nucleic acid extraction. Subsequently, use a portion of the total nucleic acid and reverse- transcribe RNA into cDNA for -20°C storage. To do this, follow the initial steps of the water sample testing SOP (REDI-NET Water Testing SOP W-4) cDNA synthesis until finishing step 40.

Each collected water sample will be given a unique ID.

Store water samples for up to 24 hours at 4°C and up to 7 days at -20°C before processing the samples.

Follow the SOP for water processing (REDI-NET SOP W-2 Water Processing) till the section “Microorganism collection” is completed.

Each water sample filtered membrane will be moved into an individual 60 mm Petri dish labeled with its original sample ID.

Seal the Petri dish with parafilm and multiple Petri dishes can be stacked together, enclosed with Saran wrap and transferred to a -80°C freezer.

Update the freezer inventory so that samples can be tracked properly.

STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID

Note

NOTES : The following procedure is to properly store total nucleic acid extracted from water samples (including negative controls) using KingFisher nucleic acid purification system. The eluted total nucleic acid will be in either 96-well microplate (Flex model) or elution strip (Duo Prime model).Total nucleic acid samples need to be kept On ice all the time to minimize RNA degradation.

In the clean PCR workstation, carefully transfer the eluted total nucleic acid to a 96-well PCR microplate.

Note

IMPORTANT : Mark the “A1” position of the 96-well microplate to prevent any mistakes on plate orientation.

Cover the 96-well PCR microplate with adhesive film to prevent spill over or contamination.

Label the film with a unique plate ID.

10.

Immediately transfer the 96-well PCR microplate to -80°C freezer.

11.

Update the freezer inventory so that samples can be tracked properly.

Disclaimer

Abstract

Steps

STORAGE PROCEDURE FOR UNTREATED SAMPLE

STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID

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