Wiese Lab:Sequencing Reaction Setup

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Revision as of 19:33, 25 February 2008

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 ' '
101.111
componentul per rxnmult factorvolume
enzyme21122
buffer31133
water1111121
final vol20176
check16

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

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