BCR 2.0
Switchable Antigen-Sensing System (S.A.S.S.)
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Tuesday
To do's
To Do's- Gel for oligo annealing
- Mix new annealing buffer
- Anneal oligos
- Design IgM Sequencing Primers
- CD79B, gal4 PCR
- Golden Gates:
- hEF1a:CD79B-pTET:mRFP-Gal4 golden gate
- TRE:Syk-15-tTEVp
- Transform
- hEF1a:CD79B-pTET:mRFP-Gal4 GG
- TRE:Syk-15-tTEVp GG
- TRE:Syk-pTET:mRFP-tTEVp sequencing
- Pick pENTR RFP-IgM Colony
Wednesday's to Dos
- rePCR Gal4, CD79B
- Gel PCRs
- GG hEF1a:CD79B-pTet:RFP-Gal4VP16
- Transform hEF1a:CD79B-pTet:RFP-Gal4VP16
Thursday's to Dos
- Review Syk-RFP-tTEV Cloning (rePCR?)(Used wrong RFP for GG?)
- hEF1a:Gal4 pick new colony
- pick hEF1a:CD79B-RFP-Gal4VP16 colonies (no red colonies present)
- pick pENTR RFP-IgM Colonies
- Innoculate midis of CDA linker
- Pick colonies of failed CDA linker constructs
- Analyze TRE:Syk-pTet:RFP-tTEV Sequencing
- Resend correct TRE:Syk-pTet:RFP-tTEV for sequencing
Friday's to Dos
- inoculate cd79a 15t3 midi
- send pENTR RFP-IgM for sequencing
- send cd79a 15t9 for sequencing
- analyze cd79a linker sequencing for previously failed linkers when it comes in
:
- Midiprep (from freezer)
- S9TEV
- B15T15
- Golden Gate
- B9T9
- B15T9
- LR
- hEF1a-CuO:Gmab H
- hEF1a-CuO:Gmab L
- hEF1a-CuO:CD79A
- hEF1a-CuO:CD79B
- hEF1a-CuO:Lyn
- Digest all of the Syk-TEVp and Syk-tTEVp midis just to be sure (since there's an internal cut site that didn't get read in sequencing)
Sequencing account information:
^^^Email: bcrpeeps@mit.edu
^^^Pass: grapefruit
## Digest tTEVp and TEVp midis just to be sure (since there's an internal cut site)
Non-Lab To Do's
- Cytometry time
- Order Cumate
- Chicken lysozyme, gp120
- Antibodies for antigen binding detection
Waiting on primers to do list:
- RFP-IgM sequencing
- Bug Brian about ordering antibody testing supplies
Higher Level To Do's:
- Decide on other antigen-variable regions we want to test
- Is gantenerumab proprietary and can we actually publish with it?