1) cells, enzymes and kits
DH5a competent cells (CB101), TIANGEN, Cat#CB101-02
DNA Loading buffer, EX-VISION, Cat#N313-1ML
DNA Ladder marker, Fermentas, Cat#SM0311
RNaseZap™ Wipes, Invitrogen, Cat#AM9786
RNeasy Micro Kit (50), QIAGEN, Cat#74004
iScript™ cDNA Synthesis Kit, Bio-Rad, Cat#1708891
AxyPrep™ PCR Cleanup Kit, Axygen, Cat#AP-PCR-250
Phusion DNA polymerase, Thermo, Cat#F-530L
La Taq, TAKARA, Cat#RR52AG
Taq 2x Master Mix, BioLabs, Cat#M0270S
Gibson Assembly® Master Mix, NEB, Cat#E2611L
5-terminal primer (5’->3’):
Up-pcDNA3.4-rab-IgLcgaacccttgctagc ccAcc ATGGACACGAGGGCCCCCACTCAG
Up-pcDNA3.4-rab-IgHtcgaacccttgctagc ccAcc ATGGAGACTGGGCTGCGCTGGCTT
rab-IgLV ATGGACACGAGGGCCCCCACTCAG
rab-IgHV ATGGAGACTGGGCTGCGCTGGCTT
3-terminal primer (5’->3’):
rab-IgL-V-p1CAGTTGTTTGGGTGGTGCCATCCAC
rab-IgH-V-p1GCTGGCTGCTTGAGGTCACGCTCACCAC
rab-IgL-V-PexTTCGCCACACACACGATGGTGACT
rab-IgH-V-Pexggcagcccagggtcaccgtggagct
3) cell expression system
Expi293™ Expression Medium, Life, Cat#A1435101
Opti-MEM, Life, 31985062, 100mL
ExpiFectamine™ 293 Transfection Kit, Life, Cat#A14524
AxyPrep™ Plasmid Kit, Axygen, Cat#AP-96-P-4ExpiFectamine™ 293 Transfection Kit, Life, A14524 (including ExpiFectamine,enhancer 1 ,enhancer 2 )
4) Buffer and solution for antibody purification
Protein A binding buffer: 25mM Tris-HCl, 25mM NaCl, pH7.2
Elution buffer: Bio-Rad Cat#1536161 , prepared following the manufacturer's instruction
Neutralizing buffer: 1M Tris-HCl, pH9.0
Thermal cycler (PCR machine),DNA electrophoresis system, 37℃ incubator,37℃ orbital shaker, TC-10 cell counter,centrifugate,pH meter,magnetic stirrer, vortex shaker.
GGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGCTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCACGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATGA
ATGGTGACCTTACCCTACTACTTTAACTTGTGGGGCCAAGGCACCCTGGTCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGCTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCATGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATGA