Template FAQ

How much plasmid DNA do you need for a standard premixed tube reaction?

• 1,000-1,500 ng ds plasmid DNA template
• 2 µl 4 µM primer
• x µl sterile water
• 18 µl total volume

How much plasmid DNA do you need for a high volume premixed 96-well reaction?

• 1,000-1,500 ng ds plasmid DNA template
• 2 µl 4 µM primer
• x µl sterile water
• 18 µl total volume

How much PCR product do you need for a standard premixed tube reaction?

• 100-300bp:40-50ng
• 300- 500bp:40-160ng
• 500bp- 1kb:500ng
• 2 µl 4 µM primer
• x µl sterile water
• 18 µl total volume

How much PCR product do you need for a high volume premixed 96-well reaction?

• 100-300bp:40-50ng
• 300- 500bp:40-160ng
• 500bp- 1kb:500ng
• 2 µl 4 µM primer
• x µl sterile water
• 18 µl total volume

Should I resuspend template in TE or H ₂O?

H₂O is definitely preferred. Sequencing reactions are very sensitive to chelating agents, such as EDTA.

How long is the average read?

Template quality will affect length of read. Average read length is 500bp, however, lengths of 650-750bp are routinely achieved. We often see lengths of 800bp-1100bp.