This section is designed to provide help to researchers and offer a variety of useful protocols collected around different lab.

You are invited to submit any protocol that has worked for you or your lab group. Just write an email to the Collection Manager

Collecting expeditions typically consist of a series of excursions to selected habitats interspersed with breaks to catalogue and process the collected material. Sometimes insects are left in killing jars until they are processed, resulting in DNA degradation. To best retain traces of Wolbachia, insects should not be held without preservative. They should be transferred as soon as possible into 95% ethanol, or frozen at the collecting site. Strategies for both are suggested below.

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This information can be seen in more details at: AMCC Field collection kits 

The AMCC will provide researchers of the FIBR Wolbachia project partner institutions with pre-labeled vials as well as dryshippers and an "electronic Field Collection Kit to facilitate the transfer of their samples at the AMCC.

Pre-labelled 1.8 ml vials, with AMCC label containing the AMCC number in both barcode and human readable format. These numbers are communicated to the researcher in an excel format spreadsheet as part of the "Electronic Field Collection Kit".

The vials can be either empty (for flash freezing in the field) or filled with buffer (when flash freezing in the field is not possible)

Electronic Field Collection kit

The electronic kit consists in:

- one excel spreadsheet template containing the AMCC numbers of the pre-labeled vials as well as template data fields from the AMCC database. For more on this, visit the AMCC electronic Field Collection kit page

Additional Field Equipment:

Dryshipper: The AMCC provides researchers with dry-shippers allowing control-rate freezing type of sample collecting in the field, as well as transfer from museum to museum, or donor to museum without ever thawing the specimens.

For more information on the dryshipper, see the AMCC Website

Specimens may be frozen at the collecting site. Dead or stunned insects should be placed in cryotubes without buffer and placed in the cold dryshipper. The nitrogen-charged dryshipper can be obtained from the  AMCC. Go to  http://research.amnh.org/amcc/dryshipper_req.html to download a request form. (To stun an insect, squeeze its thorax while it is in the net, or keep it in the killing jar until it is subdued.) Butterflies may be placed in labeled glassine envelopes and placed into the dryshipper alive. Series of unidentified organisms may be separated into morpho-species and frozen if vouchers are preserved dry or in ethanol for later identification.

Plastic tubes containing 95% ethanol should be included in the collector's field pack. 15ml and 50ml Falcon will hold ethanol without leaks or evaporation and are appropriate for short-term storage. They can be purchased from Fisher Scientific at www.fishersci.com. If large organisms, like tarantulas or horse lubber grasshoppers, are likely to be collected, 500ml Nalgene bottles should be included. NALGENE bottles are also available from Fisher. Insects can be pooled in dated, locality-labeled holding tubes, and held for a few days without sustaining damage. Kill and transfer the insects immediately to ethanol, or kill them directly in ethanol by tapping them into a wide-mouthed ethanol-filled tube. This works well for flies, beetles and bugs.

Identified specimens can be catalogued at the collection site by transferring them to permanent storage tubes. Empty or Ethanol-filled barcode-labeled AMCC cryotubes are recommended for this purpose. They can be obtained from jfstein@amnh.org at the AMCC. A limited number of tubes of a commercial preservative, RNA LATER, are also available for collection. (For more information, or to request field collecting kits, see below and visit http://research.amnh.org/amcc/). Right-in-the-rain weather-resistant field notebooks are recommended. They can be purchased from BioQuip at www.bioquip.com. If AMCC barcode-labeled tubes are used, the data can be simply referenced to the barcode number. Alternatively, electronic data records can be entered into an AMCC electronic spreadsheet supplied for this purpose. Host plant association is sometimes critical for the identification of insect species. When possible note the plant that the insect is on when collected, especially if it is feeding.

For species normally stored dry -- or that must be kept dry for identification -- like butterflies, it may be advisable to remove the genitalia at the collection site. Genitalia can be stored in ethanol or frozen (as explained below). The same reference number should be used to identify the genitalia and the rest of the insect, which may be kept for a pinned voucher. 

Ethanol preservation is not static and DNA will degrade in ethanol at room temperature over time. If a freezer is available, store the specimens at -4^oC (good) or -20^oC (better). WHEN THE SPECIMENS RETURN FROM THE FIELD THEY SHOULD BE HELD IN THE LAB FREEZER AT -20^oC until transfer to the AMCC. 

If collecting in RNAlater samples should be treated in a manner that adheres to the following guidelines, RNAlater effectively deactivates all enzymatic activity (endo and exonuclease activity) for 24 hours at 37°C, 7 days at 18-25°C, 2 weeks at 4°C and indefinitely at -20°C. However if immediate refrigeration is available use it.  When collecting very large arthropods breaking open the exoskeleton just before submerging the insect may promote greater profusion of RNAlater through the tissue.

The post-collection RNAlater method suggested by the manufacturer is to store the samples at 4°C for at least 24 hours, then pellet down the sample, remove the RNAlater and either begin the extraction or store the dry samples in freezers.  For storage at -20°C, it is recommended that excess RNALater be removed from the sample to prevent crystallization of RNAlater and damage of the sample. For storage in cryogenic freezers at the AMCC, RNA later is drained away and the sample is stored dry.

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RECOMMENDED FIELD EQUIPMENT: 

From BIOQUIP: Insect Net, Aspirator (with extra vials), Long forceps (10"), Killing Jar (or materials for manufacture), Right-in-the-rain field notebooks, glassine envelopes for Lepidoptera.

From AMCC: Barcode-labelled cryotubes, field kits, nitrogen-charged dry shipper. Cryotubes are issued in boxes capable of holding 100 tubes. Each has a barcode and human-readable label and is issued with an electronic spreadsheet that may be printed for inclusion in the collector's field kit, or used directly on a computer in the field. It is enough to write down the barcode number and the contents of the tube with locality and collection data.

From Fisher: Assorted plastic tubes capable of short-term ethanol storage without leaks or evaporation. 

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It is recommended that you accession your collected specimens at the AMCC as soon as possible (preferably directly after collection, or soon after if identification is required).

For collectors who have received an AMCC field kit, simply mail back the boxes of vials, or the dryshipper to the AMCC.

Send your electronic data file to the Collection Manager, Julie Feinstein (jfstein@amnh.org, 212 769 5663) who will acknowledge reception of your samples and data.

If you would like to send your samples, but have not requested a field kit prior to collecting, you may:

1. Request AMCC barcoded vials for you to transfer your own samples in the comfort of your own lab at your return from the field. Please remember to send the samples accompanying data electronically (excel spreadsheet). You may also request to have a dryshipper sent for the transfer of the samples from your institution to the AMCC.
2. Send the samples and their accompanying data (excel spreadsheet) in their original vials (we will transfer the samples ourselves, though it is not adviseable, as it adds time to the accessioning process into the collections)

Send your samples to the AMCC Collection Manager, Julie Feinstein (jfstein@amnh.org; 212 769 5663)

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A useful link: Rob Desalle Molecular Systematics Lab

Another useful series of links is on our Links page, under Molecular Bio techniques etc

- Lysis

- RNA Later

A Qiagen^TM product, RNA Later has so far proven to preserved the molecular quality of the sample best. However, it can only be preserved at room temperature for 24 hours, after which it must be refridgerated.

Protocol for RNA Stabilization with RNAlater RNA Stabilization Reagent (PDF version, 57 KB)