Wiese Lab:Sequencing Reaction Setup

Back to Protocols

Sequencing Reaction Setup

1. Assemble components for sequencing reaction, keep on ice:
   • template DNA - should be column-purified DNA. For example, Eppendorf Fast Mini with 2-3 ml O/N culture. #*concentration of stock DNA at 100 - 200 ng per ul (0.1 -0.2 ug/ul)
   • double the template concentration to improve signal strength.
   • sequencing primer diluted to 10 uM (10 pmol primer / ul)
   • Big Dye reagent
   • 5X Big Dye buffer
2. Combine reagents for sequencing reaction.
   • Determine amount of sterile water needed to create 20 ul final volume.
   • Add water, buffer and enzyme to reaction tubes, or add as a pool as described below.
   • Preparation of a buffer/reagent pool may be useful if doing several sequencing reactions.
   • To ensure sufficient quantity use a volume multiplication factor of 1.1 to calculate volumes needed. For example, 4 sequencing reactions using the same Big Dye buffer, enzyme and volume of water.
   • Prepare a reagent pool containing:

# samples	overage factor	mult factor	'	'
10	1.1	11
component	ul per rxn	mult factor	volume
enzyme	2	11	22
buffer	3	11	33
water	11	11	121
final vol	20		176
			check	16

3. Amplify template in thermocycler. Remember to check thermocycler settings!

'	'	'	File Number	'	'
Temp ( C )	Time	x Cycles	Cycler #1	Cycler #2	Cycler #3
95	3 min	1	55	53	26
96	10 sec	50	58	48	25
58	4 min
72	7	1	10	47	24
4	infinite		11	44	23