Gel Loading Dye, Purple (6X) (Cat #B7024S, New England Biolabs) 13. UV–Vis spectrometer (Nano-photometer N60, IMPLEN) 12. Presto™ Mini plasmid extraction kit (Cat #PDH100, Geneaid) 10.ĮasyPrep EndoFree Maxi plasmid extraction kit (Cat #DPT-BA17, TOOLS) 11. coli DH5α strain for plasmid amplication (ECOS™ 101, Yeastern Biotech) 9. PCR clean-up kit (GenepHlow Gel/PCR kit, Cat #DFH100, Geneaid) 6.Įppendorf centrifuge (Thermo Fisher Scientific) 7.Į. PCR buffer and reagents: KOD Hot Start DNA polymerase Kit (Novagen), MgSO 4 (NEB), dNTPs (NEB), HindIII (NEB), EcoRI (NEB), DpnI (NEB), NEB3.1 (NEB), T4 ligase and T4 ligase buffer (NEB) 5. Polymerase chain reaction (PCR) thermocycler (Mastercycler EP Gradient S, Eppendorf) 4. The protein sequences of variants follow the definitions in PANGOLIN database ( ). Synthetic genes corresponding to the S protein variants are codon-optimized for HEK293 expression. PcDNA™3.4 TOPO™ TA Cloning Kit (Invitrogen, Cat #14697) 2. Alternatively, the vector can be cut with restriction enzymes that generate a single T overhang. The missing hydroxyl ensures that only one T is added to the vector ends. Instead of purchasing a prepared vector, the T overhangs on the vector can be generated by first cutting the vector with a blunt end restriction enzyme, and then by mixing it with a terminal transferase enzyme and dideoxythymidine triphosphate (ddTTP), which is missing the 3′ hydroxyl group. Compatible TA cloning vectors with T overhangs are supplied by different biotechnology companies. ![]() This reaction does not depend on the sequence. This is not true for all thermostable DNA polymerases, but is true for Taq polymerase. Taq DNA polymerase has terminal transferase activity, which means it adds a single deoxyadenosine (dA) to the 3′-ends of double-stranded DNA. ![]() McGehee, in Molecular Biology (Third Edition), 2019 2.2 TA Cloning of Polymerase Chain Reaction (PCR) ProductsĪnother approach, called TA cloning, creates complementary single-stranded overhangs between the insert and vector by exploiting a secondary enzymatic property of Taq polymerase.
0 Comments
Leave a Reply. |