STATUS: COMPLETE Description:Making pEXPRs hEF1a:LilrB2 and hEF1a:PirB. | Expand |
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| Status:- First and second set of liquid cultures didn't grow, possibly because:
- Too much media
- Plates were old, antibiotic possibly not working
- 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, pENTR only has C terminus end of PirB
- New PirB pENTR colonies picked, grown and miniprepped
- Restriction digest ran - correct band pattern for one of the colonies
- Correct colony sent out for sequencing - pENTR has correct sequence
- 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
- LilrB2 pEXPR sent out for sequencing
- incomplete sequencing results
- LilrB2 insert is in the pEXPR
- LR with new PirB pENTR made
- Restriction digest and gel ran to verify LR products
- PirB insert is in the pEXPR
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| Plan:- 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 and verify product
- Make glycerol stocks
- LR into pDEST
- Transform, pick colonies and grow in liquid culture
- Miniprep cells and verify product
- Make glycerol stocks
- Midiprep product
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