Wiese Lab:Sequencing Reaction Setup
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(New page: Back to Protocols ==Sequencing Reaction Setup== #Assemble components for sequencing reaction, keep on ice: #*template DNA - should be column-purified DNA. F...)
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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||'||'|
|component||ul per rxn||mult factor||volume|
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