Wiese Lab:Sequencing Reaction Setup

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Sequencing Reaction Setup

 * 1) Assemble components for sequencing reaction, keep on ice:
 * 2) *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)
 * 3) *double the template concentration to improve signal strength.
 * 4) *sequencing primer diluted to 10 uM (10 pmol primer / ul)
 * 5) *Big Dye reagent
 * 6) *5X Big Dye buffer
 * 7) Combine reagents for sequencing reaction.
 * 8) *Determine amount of sterile water needed to create 20 ul final volume.
 * 9) *Add water, buffer and enzyme to reaction tubes, or add as a pool as described below.
 * 10) *Preparation of a buffer/reagent pool may be useful if doing several sequencing reactions.
 * 11) *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.
 * 12) *Prepare a reagent pool containing:




 * align="center" style="background:#f0f0f0;"|# samples
 * align="center" style="background:#f0f0f0;"|overage factor
 * align="center" style="background:#f0f0f0;"|mult factor
 * align="center" style="background:#f0f0f0;"|
 * align="center" style="background:#f0f0f0;"|
 * 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
 * }
 * water||11||11||121||
 * final vol||20||||176||
 * ||||||check||16
 * }
 * ||||||check||16
 * }
 * }


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


 * align="center" style="background:#f0f0f0;"|
 * align="center" style="background:#f0f0f0;"|
 * align="center" style="background:#f0f0f0;"|
 * align="center" style="background:#f0f0f0;"|File Number
 * align="center" style="background:#f0f0f0;"|
 * align="center" style="background:#f0f0f0;"|
 * 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.
 * 72||7 min||1||10||47||24
 * 4||infinite||||11||44||23
 * }
 * 4. Clean up sequencing reaction.
 * }
 * 4. Clean up sequencing reaction.