Wiese Lab:Sequencing Reaction Setup
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Current revision
Sequencing Reaction Setup
- 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
- 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 min 1 10 47 24 4 infinite 11 44 23
- 4. Clean up sequencing reaction.
