Methods for Researchers
Welcome to the Methods for eDNA Researchers page! Here you’ll find links to up-to-date protocols for our standard CALeDNA DNA library preparation workflow. In the software link above, you will find our data analysis tools Anacapa: a toolkit to process multiple metabarcode eDNA libraries, and Ranacapa: a community ecology data exploration tool.
If you find anything from a typo to a phrase that is unclear or erroneous in any of the workflow, please let us know and we’ll fix it right away! Send your comment to firstname.lastname@example.org.
CALeDNA Library Preparation
Obtain samples from the archival freezer, thaw slightly on ice, and make an aliquot pool of biological replicates.
Freezer map guide is HERE (UC RESEARCHERS: TO UPDATE PLEASE REQUEST ACCESS TO GOOGLE SHEET)
Protocol for sample pooling is HERE
Extract DNA from the aliquot of soil or sediment. If extracting DNA from water filters, stay tuned: protocols coming.
Detailed protocol for soil or sediment DNA extraction is HERE
Experienced user short protocol for soil or sediment DNA extraction is HERE
Do three half-reaction PCRs for each marker. We typically use 4-6 markers.
PCR thermocycler settings for amplifying routine markers: 16S, 18S, CO1, plant ITS2, fungal ITS, fish 12S, and trnL are HERE
Primer sequence info is HERE
Detailed protocol for PCR of markers is HERE
Experienced user short protocol for PCR of markers is HERE
Run a gel to check if your PCR worked to amplify your target markers.
Detailed protocol for gel electrophoresis and imaging is HERE
Experienced user short protocol for gel electrophoresis and imaging is HERE
Pool your three PCRs for each marker into one pool per marker per sample.
Detailed protocol for pooling PCR products is HERE
Experienced user short protocol for pooling PCR products is HERE
Purify your pooled PCR products using beads.
Detailed protocol for bead cleaning PCR products is HERE
Experienced user short protocol for bead cleaning PCR products is HERE
Use a Qubit DNA BR assay to quantify how much DNA is in your cleaned PCR products. Use the standard protocol that comes with the BR assay for quantifying one sample at a time. These protocols below are for batch quantification (>16 samples) using a plate reader.
Detailed protocol for Qubit plate quantification is HERE
Experienced user short protocol for Qubit plate quantification is HERE
Convert your Qubit fluorescence readings from the plate reader to DNA concentration in Excel. Instructions are in the protocols, and an example template is provided HERE
Use your PCR product concentrations to pool the products by sample. Follow the template guide.
Download the template to pool amplicons prior to indexing HERE.
Detailed protocol for pooling by sample HERE
Experienced user short protocol for pooling by sample HERE
Do a PCR to add indices to your samples. Make sure these are unique combinations for all samples going on the same sequencing lane. A template is provided to help you organize your indices in a plate format.
Detailed protocol for indexing PCR is HERE
Experienced user short protocol for indexing PCR is HERE
Template for organizing indices is HERE
Run a gel with 5uL of your indexed product to make sure the reaction worked. Bands should be ~40bp larger than the size of the original marker PCR. Because you are indexing multiple marker amplicons, you may see a smear of DNA spanning a broad size range. Look out for high molecular weight DNA. If you see anything over 1kb, you will need to do size selection (protocols not included).
Use the templates from Step 4.
Purify your indexed PCR products.
Use the templates from Step 6.
Quantify your cleaned indexed PCR products. Use the standard protocol that comes with the BR assay for quantifying one sample at a time. These protocols below are for batch quantification (>16 samples) using a plate reader.
Use the templates from Step 7.
Pool your cleaned indexed PCRs (i.e. your sample libraries) by even numbers of molecules per sample. Note: you may decide that blanks/negative controls don’t need to be even with the real samples. Use the the concentration you calculated in Step 12 and the estimated size of the bands or smears in your gel in the template provided to make the calculations. Put the pooled samples in to a special Lo-Bind microcentrifuge tube.
Detailed protocol for pooling libraries HERE
Experienced user short protocol for pooling libraries HERE
Template for pooling libraries is HERE
Store Lo-Bind tubes of libraries at 4 degrees C for up to 5 days prior to sequencing. Freeze pooled libraries at -20 degrees C for longer storage. We recommend you split your pooled library into two tubes: one goes to the sequencing facility and one stays with you as a back up.
We have been submitting libraries for sequencing to these fine facilities:
UC Berkeley Vincent Coates Genome Sequencing Laboratory http://qb3.berkeley.edu/gsl/
UCLA Technology Center for Genomics and Bioinformatics http://pathology.ucla.edu/tcgb