laboratory

ddRADSeq Library Prep

• DNA Extraction
• Quantify
• Digest
• Clean
• Quantify
• Anneal adapters
• Dilute adapters
• Robot
• Ligate DNA
• Pool
• Clean 1
• Clean 2
• Size Select
• PCR
• Baits
• Sequencing Prep

WARNING: These protocols are abridged versions of the manufacturers recommended protocols. Use of these protocols assumes familiarity with manufacutrers instructions. Please read manufacturers instructions if this is your first time (or first time in a while) performing these procedures.

DNA Extraction

Procedure
Lab Notebook

• Takes 24 hours, 16 hours of overnight lysis

Quantify

Procedure Lab Notebook

• Takes 0.5 hours

Digest

Procedure Lab Notebook

• Takes 3 hours add 1/2 hour for each additional plate

Clean

Procedure Lab Notebook

• Takes 1 hour per plate

Quantify

Procedure Lab Notebook

• Takes 1/2 hour for each plate or qubit run

Anneal adapters

Procedure Lab Notebook

• Takes 1 hour, 1/2 hour in thermocycler

Dilute adapters

Procedure Lab Notebook

• Takes 1/2 hour

Robot - because of the large quantity of samples (over 1000), we used a pipeting robot to set up the ligation plates

Procedure Lab Notebook - Takes 1/2 hour per plate plus travel to and from Princeton
- Quality control after robot

Ligate DNA

Procedure Lab Notebook

• Takes 4 hours plus 1/2 hour per plate

Pool

Procedure Lab Notebook

• Takes 1 hour

Clean 1

Procedure Lab Notebook

• Takes 1/2 hour

Clean 2

Procedure Lab Notebook

• Takes 1/2 hour
• Elute into 30 ul

Size Select

Procedure Lab Notebook

• Takes 2 hours per cartridge (5 pools per cartridge)

PCR

Procedure Lab Notebook

• Takes 2 hours

Baits

Procedure
Lab Notebook - Takes 2 days

Sequencing Prep

Procedure Lab Notebook

• Takes 1/2 hour