Tuesday'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, 15t15, 9t9 midis
- analyze cd79a linker sequencing for previously failed linkers when it comes in
- Golden Gate Syk-linker-tTEV linkers and transform gg
- Re-PCR Q3-RFP-Q4
- Transform:
- GG hEF1a:CD79B-pTet:RFP-Gal4VP16 with new RFP
- TRE:Syk-linker-tTEVp golden gates
- Gel:
- digests
- aari diagnostic
- Sequencing
- cd79a linkers
- pENTR RFP-IgM
- Miniprep 3t15, 15t9, hef1a:gal4
- midi linkers
- Digest minis and midis
Monday's to Dos
- Midiprep
- something
- hEF1a:CD79B-pTet:RFP-Gal4VP16
- ?
- Pick Colonies
- TRE:Syk-3-tTEV
- TRE:Syk-6-tTEV
- TRE:Syk-9-tTEV
- TRE:Syk-12-tTEV
- TRE:Syk-15-tTEV
Sequencing account information:
^^^Email: bcrpeeps@mit.edu
^^^Pass: grapefruit
Non-Lab To Do's
- Cytometry time
- Order Cumate
- Chicken lysozyme, gp120
- Antibodies for antigen binding detection
Waiting on primers to do list:
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?
THINGS TO NOT FORGET TO DO:
SYK-15tTEVP golden gate when we figure out what's wrong with the AarI