Cloning Strategy for IgM heavy and light chains (outdated?)
Order G blocks "mIgM heavy chain against beta amyloid" and "mIgM kappa chain against beta amyloid" that are flanked with complementary sequences to Nelson's primer with Q1 and Qx sites
PCR amplify G blocks with Nelson's primers
Golden gate G blocks into a pDONR vector creating pENTR L1-mIgMheavy-L2 and pENTR L1-mIgMkappa-L2
Transform bacteria and plate on kan+ media
Miniprep out pENTR L1-mIgMheavy-L2 and pENTR L1-mIgMkappa-L2 vectors
LR pENTR L1-mIgMheavy-L2 and pENTR L1-mIgMkappa-L2 vectors each with pENTR L4-Hef1a-R1 to create pEXPR hef1a-mIgMheavy and pEXPR hef1a-mIgMkappa for expression of the antibody
Transform bacteria again?
PCRs to do:
| Product | Gene | Starting Plasmid | Forward Primer | Reverse Primer | Tm hi/lo (anneal) | PCR's that can go together |
|---|---|---|---|---|---|---|
| CD79A | CD79A | pENTR CD79A (pDONR223) | iGEM KRB 003 F | iGEM KRB 004 R | 62/61 (61) | 1 |
| CD79A-TCS | CD79A | pENTR CD79A (pDONR223) | iGEM KRB 003 F | iGEM KRB 005 R | 62/61 (61) | 1 |
| CD79B | CD79B | pENTR CD79B (pDONR223) | iGEM_AS001 CD79B Q1_F | iGEM_AS002 CD79B QX_stop_R | 72/70 (70) | |
| CD79B-TCS | CD79B | pENTR CD79B (pDONR223) | iGEM_AS001 CD79B Q1_F | iGEM_AS003 CD79B Qlinker_R | 72/70 (70) | |
| TCS-Gal4VP16 | Gal4VP16 | pEXPR 5 – hEF1a-FGFR-mKate-TCS(L)-G4 | iGEM KRB 001 F | iGEM KRB 002 R | 62/60 (63) | |
| Lyn | Lyn | pENTR Lyn (pDONR223) | iGEM CR 006 F | iGEM CR 007 R | 61/60 (60) | 1 |
| Lyn-mKate | Lyn | pENTR Lyn (pDONR223) | iGEM CR 006 F | iGEM KRB 011 R | 61/60 (60) | 1 |
| Lyn-(15aa) | Lyn | pENTR Lyn (pDONR223) | iGEM CR 006 F | iGEM_AS009 Lyn Qlonglink_R | 61/60 (60) | |
| mKate | mKate | pENTR_L1_mKate_L2 | iGEM KRB 006 F | iGEM KRB 007 R | 58/61 (58) | 1 |
| Syk | Syk | pENTR Syk (pDONR223) | iGEM_AS004 Syk Q1_F | iGEM_AS005 Syk QX_stop_R | 54/54 (54) | |
| Syk-eYFP | Syk | pENTR Syk (pDONR223) | iGEM_AS004 Syk Q1_F | iGEM KRB 010 R | 54/54 (54) | |
| Syk-(15aa) | Syk | pENTR Syk (pDONR223) | iGEM_AS006 Syk Q1_F | iGEM_AS007 Syk Qlonglink_R | 61/59 (59) | |
| eYFP | eYFP | pENTR_L1_EYFP_L2 | iGEM KRB 008 F | iGEM KRB 009 R | 59/57 (57) | |
| (15aa)-TEVp | TEVp | pENTR_L1_Tev2 | iGEM_AS010 TEVp Qlonglink_F | iGEM_AS011 TEVp QX_R | 60/60 (60) |