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The dna2Δ pif1Δ mre11-D56N and dna2Δ pif1Δ mre11-H125N clones were as X-ray sensitive as the mre11Δ pif1Δ strain (compare Figure 3A and Figure 3B to Figure 1).
Bacterial periplasmic expression and analysis (ELISA and biosensor binding) of 12Y-2 clones were as outlined previously [ 23].
ESC cultures, electroporation, and mini-Southern-blot analysis of ESC clones were as described previously (Ramírez-Solis et al., 1993).
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The sequences of the primer sets that were used to construct the genomic clones are as follows.
The distribution of the expressed clones was as follows; 35% bovine clones, 31% mouse clones and 34% Xenopus laevis clones.
The steps in cloning are as follows.
With synthetic gene services, molecular cloning is as easy as ordering a pizza.
This simple analysis revealed that none of the clones was as a result of in vitro PCR recombination.
Secondary PCR reactions and cloning were as described above, generating pWSKDE-2HA and pWSKDE-KR-2HA.
Primers used to amplify the peptide insert for cloning were as follows: Forward 5′-TTCCGTAGCTTGTACATGTGGCCAG-3′ and Reverse ′-CACCGCTGCCACCGCT-3′.
Primers used for silent mutant cloning were as follows: Forward primer: 5′-GGGAAGTTCTACGTATGCGAGGTACTATCTCCCGTGAACACCC-3′ and reverse primer: 5′-GGGTGTTCACGGGAGATAGTACCTCGCATACGTAGAACTTCCC-3′.
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