In order to further investigate the role of the c-myb intron in expression, we attempted to amplify the Myb-INT gene fragment. Myb-INT is a 4500 bp DNA fragment from the first c-myb intron, and has a GC content of about 60%. Sequences that contain a high ratio of guanosine and cytidine base pairs (GC-rich sequences) can form robust secondary structures that resist denaturation and prevent annealing of PCR primers.
PCR was performed using HotStarTaq DNA Polymerase and 4 other commercially available DNA polymerases. Of the 5 polymerases tested, only HotStarTaq DNA Polymerase (in conjunction with Q-Sol ution) allowed successful amplification. After amplification, the fragment was successfully subcloned into the pDrive Cloning Vector using the QIAGEN PCR Cloningplus Kit and transformed into QIAGEN EZ Competent Cells.Materials and methods
Several primer pairs were designed to amplify a 4.5 kb Myb-INT fragment
of the c-myb gene. The PCR template was plasmid DNA purified using the
Maxi Kit. Each PCR contained 300 ng template DNA, 2.5 mM MgCl2, 0.2
mM each dNTP, and 2.5 units DNA polymerase, in 1x PCR Buffer. PCR was
carried out in the presence and absence of 1x Q-Solution. PCR products
were purified using the MinElute
PCR Purification Kit. For the cloning procedure, 225 or 550 ng purified
PCR product was added to 50 ng pDrive Cloning Vector in 1x Ligation Master
Mix, in a total volume of 10 l. Subsequently, a 3 l aliquot
of the ligation reaction was used to transform one tube of QIAGEN EZ Competent
Cells, which were then directly plated onto LB agar plates containing
X-gal and IPTG for blue/white screening, and ampicillin at 30 g/ml.
A second set of ligation reactions were carried out, and 3 l aliquots
used to transform electrocompetent DH5-a E. coli. After transformation,
bacteria were grown at 37C for 40 minutes before plating as above.
White colonies derived from both ligation reactions were picked and grown
overnight at 37C in LB medium containing ampicillin at 20 g/ml.
Pure plasmid DNA containing the PCR insert was isolated using the QIAprep
Spin Miniprep Kit. The presence and correct orientation of the insert
was confirmed by restriction digest using ClaI and SacI and by direct
Successful PCR Amplification of a GC-Rich Gene Fragment
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.< /p>
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).