( %20_art.htmlQIAprep Spin M...)Successful PCR amplification and subcloning of a GC-rich DNA fragment

%20_art.html">QIAprep Spin Miniprep Kit. The presence and correct orientation of the insert was confirmed by restriction digest using ClaI and SacI and by direct sequencing.

Successful PCR Amplification of a GC-Rich Gene Fragment

PCR of a GC-rich c-myb gene fragment using HotStarTaq DNA Polymerase (QIAGEN) or DNA polymerase from other sources (Suppliers WZ) in the presence (+) or absence () of Q-Solution. M: markers.

Proportion of recombinants containing c-myb gene PCR insert after transformation with pDrive Cloning Vector

E.coli strain % white colonies

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QIAGEN EZ Competent Cells 90% DH5-a cells 88%
Results and discussion

The efficiency of amplification of the 4.5 kb PCR product using the five different DNA polymerases is shown in Figure "Successful PCR Amplification of a GC-Rich Gene Fragment". Only the reaction containing HotStarTaq DNA Polymerase and Q-Solution enabled amplification of the GC-rich c-myb gene fragment. Q-Solution facilitates amplification of difficult templates by modifying the melting behavior of DNA.

An aliquot of the PCR product was used to successfully clone the c-myb gene fragment into the pDrive Cloning Vector. Cloning into this vector is based on hybridization of 3'-end A overhangs (added to PCR products by HotStarTaq DNA Polymerase) to U overhangs on each end of the linearized pDrive vector.<
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