Notebook Timeline
Welcome to our notebook! As you scroll down the timeline you can see our actual notes for what we did each day.
For the 2A9N - remove the H and the L chains from sequence (only want the M and I).
Need to do:
- Make the 9 Bsa1 sites
- Check NEB digest - get above 90% chance of working/not binding with the wrong overhang
- Check methylation sites
Bsa1 cutting sites--9 regions and 9 cutting sites
Region 1 - linker region 1 (ScFv1 and AraC1)
Region 2 - AraC1 and ribo linker and ScFv2
Region 3 - Linker region 2 (ScFv2 and AraC2)
Region 4 - AraC2 and part of plasmid (1)
Region 5 - Plasmid (1) (Plasmid 1 w/ enzymes)
Region 6 - Plasmid (2) to Plasmid (3) (Plasmid 2 w/ enzymes)
Region 7 - Plasmid (3) and Plasmid (4) (Plasmid 3 w/ enzymes)
Region 8 - Plasmid (4) and Plasmid (4) with the ScFv1 (Plasmid 4 with enzymes)
Region 9 - Plasmid (4) with the ScFv1
From Twist: 100 ng - 1 μg for each fragment
Fragments were ordered today.. expect to recieve them by June 12th. Next order by date is June 12 - get by July 10th
Chain M (Scfv2) in the structure of Fluro antibody missing residue 149 (Gly)
Continued to work on modeling and grants.
A -> C = Scfv1 + Arac1. B -> D = Scfv2 + Arac2
Continued modeling and fixing different linkers
***Linker additions made to DNA fragments on 6/3 changed scfv1=A, scfv2=C, arac1=B, arac2=D on pymol files
For control, IgG1 ~ 1ngq antibody - humanized
Received DNA from Twist, will bring up DNA to create stock solutions tomorrow.
TEST
DNA IN EACH TUBE:
AMOUNT OF Tris TO ADD TO BRING UP TWIST DNA:
Each piece of our DNA plasmid should now have a concentration 40 fmol/1μl
ADDITION OF AMP TO AGAR MIX:
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