Cloning of PCR product using pCRScript SK(+)Amp

• This is a good method. But if you are in a lab like me wanting to save some money, you can use my favorite one tube PCR cloning method which works equally well.
  1. Purify the PCR product using S-300 spin column (Pharmacia

  ).

• Polishing using Stratagene's PCR polishing kit:
• 10ul Pure PCR product
• 1ul 2.5mM dNTP
• 1.3ul 10X polishing buffer
• 1ul cloned Pfu polymerase
• 65^oC 30' => 70 ^oC 10'
• Ligation using Stratagene's PCR cloning kit:
• 1ul precut pCRScript SK(+) vector
• 1ul 10X reaction buffer
• 0.5ul 10mM rATP
• 4ul polished PCR product
• 1ul SrfI restriction enzyme
• 1ul T4 DNA ligase
• 1.5ul Water
• RT for 2 hrs
• 65 ^oC inactive the reaction 10'
• Add 5ul to 200ul DH5a competent cells, ice 5' and plate onto pre-warmed (37 ^oC) plates (LB + 100mg/L Amp and X-gal), 37 ^oC overnight.

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