A barcode is a 650 base pair long sequence from the COI region in the mitochondria. There are three basic parts to barcoding. The first is data submission so that all the information relating to a specimen is entered into the Barcode of Life Database (BOLD). The second is the actual lab work where you go from having tissue to the sequencing reactions. The final part of barcoding is the analysis of the data where you edit the sequences and put them onto BOLD.
The first step in DNA barcoding is the data submissions. Before you can do any lab work, you need to create a BOLD project (unless one already exists, in which case you must be added to the project), add the specimen information into BOLD in a BOLD Specimen Spreadsheet, upload the plate or box records to LIMS, and put the plates into Dataserv.
- Create a project on BOLD you can skip this step if a project already exists for the samples you are working on.
- BOLD Specimen Spreadsheet have to be filled out and submitted for the information to be on BOLD, before you can use LIMS.
- Plate Records or Box Records are necessary for LIMS submission of the data.
- The Access System (Dataserv) must be used if you are doing any steps in BIO.
Protocols for Lab Work
Once all the data has been entered to LIMS, BOLD and Dataserv, you can do lab work. The first step is tissue lysis, then extraction is either robotic in BIO or manual in the Science Complex. When you have DNA, you then have to run PCR reactions, e-gel them, put all the steps on LIMS, and then sequence the samples.
- Tissue Lysis protocols for BIO but also instructions on subsampling.
- Robotic DNA Extraction in BIO
- Manual Extraction done in the Science Complex.
- FTA Cards may be sent rather than tissue.
- Making PCR plates for BIO if you do not have pre-made plates or the Genomics Facility.
- PCR Procedure for BIO or the Genomics Facility.
- E-gel PCR Product Check for BIO or the Genomics Facility.
- E-gel image on the Computer for BIO or the Genomics Facility.
- What to do when your Plates Fail for help trouble shooting.
- Using the Nano Drop
- Making Sequencing Plates for BIO Protocols
- Sequencing Procedure For BIO
- Sequencing at the Genomics Facility
Analysis of Data
After you have completed all the data submissions and lab work, you can finally analyze the resulting sequences. The sequences have to be edited with a sequence editing program. There are many different programs, but in the Hanner lab we have SeqScape and Sequencher. Once the sequences have been edited, the sequences and trace files have to be uploaded to BOLD.
- Sequence Editing with SeqScape
- Sequence Editing with Sequencher
- Sequence Editing with CodonCode Aligner coming soon!
- Trace File Submission necessessary for all sequences that are put onto BOLD.
- Problems with sequences for trouble shooting help.
Ordering, Mailing, and other Miscellaneous Things to Know
This is a list of miscellaneous things that should help with lab work, such as ordering supplies from various places and how to mail things to people.
- Ordering Supplies from Chem Store
- Ordering Supplies from BIOBAR
- Getting Supplies from BIO
- Ordering Supplies from Other Sources
- Mailing with Dry Ice
- How to get a LIMS Card
Created April 2009