( /p Aliquots of the ligation r...)Successful PCR amplification and subcloning of a GC-rich DNA fragment

Aliquots of the ligation reaction were used to efficiently transform DH5-a and QIAGEN EZ Competent Cells (see Table "Proportion of recombinants containing c-myb gene PCR insert after transformation with pDrive Cloning Vector"). Restriction enzyme analysis and direct sequencing confirmed that all white colonies obtained after plating contained the PCR insert (data not shown).

Conclusions

• Of 5 polymerases tested in PCR, only HotStarTaq DNA Polymerase and Q-Solution enabled successful amplification of a "difficult" GC-rich template.
  
  
• The QIAGEN PCR Cloningplus Kit enabled quick and efficient subcloning of the amplified insert into the pDrive Cloning Vector.
  
  
• After transformation, a high proportion of transformants contained the desired insert. The short ligation incubation time and the ability to immediately plate transformants using the QIAGEN PCR Cloningplus Kit streamlines cloning and screening procedures.

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