Cloning Strategy for IgM heavy and light chains (outdated?)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?
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 |