Broken constructs
Fix the Things:
hEF1a:Gal4VP16
Status:
All minis look good - all midis have extra band.
Solution:
Digest with different enzyme
Transfect and see if it works
07/29:
hEF1a:CD79B-RFP-G4
Status:
All AarI golden gates give no red colonies. We suspect bad AarI - we have new enzyme.
Solutions:
Try new AarI enzyme
Try old school restriction enzyme cloning
07/29:
Try AarI golden gate (CIDAR - 25 cycles, 40fmol each part, transform 4uL)
TRE:Syk-X-tTEVp
Status:
All BsaI golden gates give all white colonies (sketchily efficient). When digested -> no bands? lol. We have no idea what’s wrong.
Solutions:
Try again - because the whole thing was weird from the beginning.
07/29:
Try BsaI golden gate (CIDAR - 25 cycles, 40fmol each part) with extra ligation after the protocol
TRE:Syk-15-tTEVp
Status:
We keep AarI golden gating into a pEXPR which doesn’t offer us any way to screen successful reactions.
Solutions:
Golden gate with new AarI into a pENTR.
07/29:
Try AarI golden gate (CIDAR - 25 cycles, 40fmol each part)