...
- Order LilrB2 and PirB gBlocks
- Simulate Golden Gate on Geneious and make sure it works
- Golden Gate gBlocks into ggDONR
- Transform bacteria with Golden Gate product and plate
- Pick colonies and grow in liquid culture
- Miniprep cells
- Verify miniprep product
- LR into pDEST
- Transform, pick colonies and grow in liquid culture
- Miniprep product
- Midiprep product
- Transfect into HEK293
Status:
- First and second set of liquid cultures didn't grow, possibly because:
- Too much media
- Plates
were - First and second set of liquid cultures didn't grow, possibly because:
- w
- First set of plates didn't have any blue colonies
- Second set of plates grew blue and white colonies
- PirB didn't grow any white colonies
- Colonies picked for LilrB2
- Liquid culture grew
- Miniprepped - Product to be verified
- Golden Gate repeated
- cells transformed and plated
- +ve control didn't grow
- LilrB2 grew blue and white colonies
- PirB grew white colonies
- Both liquid cultures grew
- Miniprepped - Product to be verified
- pENTRs sent for sequencing - waiting on results
- LilrB2 pENTR is correct
- PirB pENTR not correct - Q-Q ligation ligation, pENTR only has C terminus end of PirB
- New PirB pENTR colonies picked
- LRs with pENTRs made
- Restriction digest and gel ran to verify LR products
- LilrB2 gave inconclusive results
- possible incomplete digestion
- PirB results did not match expected band pattern
- Due to incorrect pENTR
- LilrB2 gave inconclusive results
- Restriction digest repeated for LilrB2
- Restriction digest and gel ran to verify LR products
Experiment A2
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- Transfect HEK293 with hEF1a:PirB/LilrB2
- Check for membrane localization using anti-PirB/LilrB2 primary antibodies and secondary antibodies conjugated to fluorphores
Status:
...
- Anti-LilrB2 and anti-LilrB2 antibodies are not verified for immunostaining
- because they are not verified, we can get them at a generous discount provided that we let abcam know of our results
- we can get an anti-(_)FP antibody and stain for the receptor fused to a fluorescent protein instead
- pursue both simultaneously?