Silver:Restriction digest protocol

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Solutions/reagents:

Parameters:

  • V - Volume of PCR product DNA obtained (µl).
  • C - Concentration of BioBrick vector (µg/µl).

Equipment:

  • Incubator
  • Reaction tubes

Steps:

  1. Digestion of PCR product
    Use the following table as a checklist for preparing the reaction in reaction tube (1):

     PCR product DNA10X BSA10X restriction bufferrestriction enzyme1restriction enzyme2distilled H2O
    Restriction Digest - PCR productV3.5 µl3.5 µl0.2 µl0.2 µlMake up the volume to 35 µl
    Note: keep the glycerol concentration below 5%, the volume of both restriction enzymes added should not exceed 5% of the total reaction volume.
  2. Incubate at 37°C for at least 1 hr.
    Incubation overnight is better.
  3. Purify digested insert using PCR product purification kit.
  4. Digestion of BioBrick vector: Recommended procedure
    Use the following table as a checklist for preparing the reaction in reaction tube (2):

     BioBrick vector10X BSA10X restriction bufferrestriction enzyme1restriction enzyme2distilled H2O
    Restriction Digest - vector7/C1 µl1 µl0.2 µl0.2 µlMake up the volume to 10 µl
    Note: keep the glycerol concentration below 5%, the volume of both restriction enzymes added should not exceed 5% of the total reaction volume.
  5. Incubate at 37°C for 12 hrs(overnight).
  6. Measure out 0.1 µl of CIP into reaction tube (2).
    Incubate at 37°C for 1 hr.

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 14 hrs, 0 mins

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