Cloning Strategy for IgM heavy and light chains (outdated?)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 | Primers | Tm |
|---|---|---|---|
| CD79A | |||
| CD79A-TCS | |||
| CD79B | |||
| CD79B-TCS | |||