The ssDNA megaprimer, generated from a method described above, was used in a second PCR to complete the cloning reaction. The second PCR consisted of (i) 0.5 µl reverse primer (100 µM), (ii) 2.0 µl of the first PCR product containing the megaprimer, (ii) cloning plasmid (~50 ng/µl), (iv) 12.5 µl Q5® High‐Fidelity 2X Master Mix (New England Biolabs), and (v) molecular biology grade water to 25 µl. A standard PCR program was run for 30 cycles. The newly amplified plasmid was recovered using a GeneJET™ PCR purification kit (ThermoFisher). The final product is a dsDNA plasmid with two nicks (one on each strand). It was then transformed into competent E. coli, which seal the nicks with DNA repair enzymes. In some instances, where only one of the chimeric primers has an overlap with the plasmid (Case Study 2), the final product is linear. An optional ligation step with T4 DNA ligase (New England Biolabs) was used here. DpnI treatment (New England Biolabs), according to the manufacturer's protocol, was used to digest original plasmid templates and improve cloning efficiency.
Dpni treatment
Manufactured by New England Biolabs
DpnI is a restriction enzyme that specifically cleaves DNA at 5'-Gm6ATC-3' recognition sequences. It is used to remove template DNA in site-directed mutagenesis experiments.
Automatically generated - may contain errors
Lab products found in correlation
T4 dna ligase, by New England Biolabs (2 mentions)
Genejet pcr purification kit, by Thermo Fisher Scientific (1 mentions)
Q5 high fidelity 2x master mix, by New England Biolabs (1 mentions)
Pflag cmv 2, by Merck Group (1 mentions)
Accuprime pfx dna polymerase, by Thermo Fisher Scientific (1 mentions)
Kod hot start dna polymerase, by Merck Group (1 mentions)
Genephlow gel pcr kit, by Geneaid (1 mentions)
Presto mini plasmid kit, by Geneaid (1 mentions)
T4 polynucleotide kinase, by New England Biolabs (1 mentions)
3 protocols using dpni treatment
1
Megaprimer-based Plasmid Cloning
2
Cloning and Mutagenesis of IKZF3 Gene
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Human AIOLOS (IKZF3) ORF clone (pcDNA3.1/C-(k)DYK-IKZF3, NM_012481) was purchased from GenScript and subcloned into the mammalian expression vector pFlag-CMV2 (Sigma-Aldrich) and pcDNA3-HA using KpnI/XbaI and EcoRI/XhoI sites, respectively. Indicated mutants for the IKZF3 were generated either by the 2 step PCR method or by the site-directed mutagenesis protocol using AccuPrime Pfx DNA Polymerase (Life technologies), followed by DpnI treatment (New England Biolabs). pCMV6-AC-Myc-DDK-Human IKZF1 (IKAROS, NM_006060) and pEGFP-C1-Sumo1 (NM_003352) used in this study were previously described (12 (link), 15 (link)). pCMV3-C-HA-human HDAC1 (NM_004964.2) and pCMV3-GFPSpark-Sumo2 (BC016775) were purchased from Sino Biological. pCMV6-Entry-IKZF2 (HELIOS, NM_016260) was purchased from Origene. pcDNA3.1/C-(k)DYK-Ubiquitin B was purchased from GenScript (UBB, NM_001281720). The detailed primer sequence information is presented in Supplemental Table 6 .
3
Site-directed mutagenesis of ALD4 gene
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pFA6a-ALD4-GFP-kanMX6, constructed in previous study (Noree, 2018 (link)), was used as a DNA template to introduce N192D, S269A, E290K and C324A mutations to the ALD4 coding sequence within the plasmid. All primers used for mutagenesis are listed in Table S1 . Primers were first phosphorylated at their 5′ ends with T4 Polynucleotide Kinase (New England Biolabs). PCR-based site-directed mutagenesis reactions were set up using KOD Hot Start DNA Polymerase (Merck). The plasmid template was removed by DpnI treatment (New England Biolabs). The mutagenized PCR products were purified using GenepHlow™ Gel/PCR Kit (Geneaid). Ligations were set up using T4 DNA Ligase (New England Biolabs) to make mutagenized PCR products (linear plasmids) become circular prior to bacterial transformation. After transformation using heat shock method, plasmids were isolated from the selected transformants using Presto™ Mini Plasmid Kit (Geneaid), and were then verified by DNA sequencing (Macrogen, South Korea). The resulting plasmids after mutagenesis were referred to as pFA6a-ALD4(N192D)-GFP-kanMX6, pFA6a-ALD4(S269A)-GFP-kanMX6, pFA6a-ALD4(E290K)-GFP-kanMX6, and pFA6a-ALD4(C324A)-GFP-kanMX6, respectively.
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