Cloning Strategy for IgM heavy and light chains (outdated?)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
Antibody Entry Vector
Take G Blocks with Q1 and QX sites
PCR amplify G blocks with respective Nelson's primers
Golden Gate G blocks into pGDONR Vectorgate 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?
AS-X127 pGGDonr-ENTR-L1-LacZa-L2 has kanamycin resistance.
| Product | Status | Gene | Starting Plasmid | Starting Plasmid Resistance | Forward Primer | Reverse Primer | Tm hi/lo (anneal) | PCR's that can go together |
|---|---|---|---|---|---|---|---|---|
| CD79A | complete | CD79A | pENTR CD79A (pDONR223) | spectinomycin | iGEM KRB 003 F | iGEM KRB 004 R | 62/61 (61) | 1 |
| CD79A-TCS | complete | CD79A | pENTR CD79A (pDONR223) | spectinomycin | iGEM KRB 003 F | iGEM KRB 005 R | 62/61 (61) | 1 |
| CD79B | new primers ordered | CD79B | pENTR CD79B (pDONR223) | spectinomycin | iGEM_AS001 CD79B Q1_F | iGEM_AS002 CD79B QX_stop_R | 72/70 (70) | 4 |
| CD79B-TCS | new primers ordered | CD79B | pENTR CD79B (pDONR223) | spectinomycin | iGEM_AS001 CD79B Q1_F | iGEM_AS003 CD79B Qlinker_R | 72/70 (70) | 4 |
| TCS-Gal4VP16 | complete | Gal4VP16 | pEXPR 5 – hEF1a-FGFR-mKate-TCS(L)-G4 | ampicillin | iGEM KRB 001 F | iGEM KRB 002 R | 62/60 (63) | 6 |
| Lyn | need to redo | Lyn | pENTR Lyn (pDONR223) | spectinomycin | iGEM CR 006 F | iGEM CR 007 R | 61/60 (60) | 2 |
| Lyn-mKate | complete | Lyn | pENTR Lyn (pDONR223) | spectinomycin | iGEM CR 006 F | iGEM KRB 011 R | 61/60 (60) | 2 |
| Lyn-(15aa) | complete | Lyn | pENTR Lyn (pDONR223) | spectinomycin | iGEM CR 006 F | iGEM_AS009 Lyn Qlonglink_R | 61/60 (60) | 2 |
| mKate | DpnI digest complete | mKate | pENTR_L1_mKate_L2 | kanamycin | iGEM KRB 006 F | iGEM KRB 007 R | 58/61 (58) | 1 |
| Syk | complete | Syk | pENTR Syk (pDONR223) | spectinomycin | iGEM_AS004 Syk Q1_F | iGEM_AS005 Syk QX_stop_R | 54/54 (54) | 3 |
| Syk-eYFP | complete | Syk | pENTR Syk (pDONR223) | spectinomycin | iGEM_AS004 Syk Q1_F | iGEM KRB 010 R | 54/54 (54) | 3 |
| Syk-(15aa) | need to redo | Syk | pENTR Syk (pDONR223) | spectinomycin | iGEM_AS006 Syk Q1_F | iGEM_AS007 Syk Qlonglink_R | 61/59 (59) | 2 |
| eYFP | DpnI digest complete | eYFP | pENTR_L1_EYFP_L2 | kanamycin | iGEM KRB 008 F | iGEM KRB 009 R | 59/57 (57) | 5 |
| (15aa)-TEVp | needs DpnI digest | TEVp | pENTR_L1_Tev2 | kanamycin | iGEM_AS010 TEVp Qlonglink_F | iGEM_AS011 TEVp QX_R | 60/60 (60) | 2 |
| Product | Status | DNA | amounts | water |
|---|---|---|---|---|
| pENTER CD79A+stop | In Progress | CD79A+STOP | .917 | 12.283 |
| pENTR CD79A-TCS-Gal4VP16 | In Progress | CD79A-TCS TCS-Gal4VP16 | .933 2.262 | 10.005 |
| pENTER Syk+STOP | In Progress | Syk+STOP | 1.029 | 12.171 |
| pENTR CD79B-TCS-Gal4VP16 | Waiting on parts | TCS- Gal4VP16 CD79B-TCS. | ||
| pENTR CD79B+STOP | Waiting on parts | CD79B+STOP | ||
| pENTR Gmab Heavy | Waiting on parts | Gmab Heavy | ||
| pENTR Gmab Light | Waiting on parts | Gmab Light | ||
| pENTR Syk-eYFP | Waiting on parts | ...eYFP Syk... | ||
| pENTR Syk-TEV | Waiting on parts | Syk... ...TEV | ||
| pENTR Lyn-TEV | Waiting on parts | ...TEV Lyn... | ||
| pENTR Lyn+STOP | Waiting on parts | Lyn+STOP | ||
| pENTR Lyn-mKate | Waiting on parts | Lyn... ...mKate |