Difference between revisions of "Wiese Lab:Sequencing Reaction Setup"

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#*Prepare a reagent pool containing:
 
#*Prepare a reagent pool containing:
  
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3. Amplify template in thermocycler.  Remember to check thermocycler settings!
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3. Amplify template in thermocycler.  Remember to check thermocycler settings!
 
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4. Clean up sequencing reaction.

Revision as of 15:40, 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 ' '
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.