hEF1a:CD79A-XnTCSXc-Gal4VP16

 06/17

• annealed linker oligos
• golden gate into RFP construct 

06/18

• transformed golden gate product

06/19

• all linkers grew colonies except 9TCS6 
• picked colonies for all except 9TCS6
• retransformed 9TCS6

06/23 

• digest and gel verification 
• 3TCS3, 3TCS9, 9TCSX show correct band pattern

06/24

• sent 9TCSX for sequencing - internal BsaI cut site in Gal4VP16 caused failed golden gates -> SDM to mutate out internal cut site

07/14

• golden gates into mutated hEF1a:CD79A-pTET:mRFP-Gal4VP16

07/15

• transformed golden gate reactions – no colonies

07/16

• replate transformations - no colonies

07/17

• redo golden gates with extra ligation step,
• transform reactions

07/19

• innoculate liquid cultures

07/20

• miniprep, digest, gel - all appear correct 

07/21

• sequence all linkers 

07/23

• Correct linkers - innoculate midis:
  • 3t9
  • 6t6
  • 6t12
  • 9t3
  • 9t6
  • 12t6
• Ambiguous sequencing - repeat + innoculate minis:
  • 9T9
  • 9T12 
  • 15T15 
• Failed linkers - innoculate minis, digest, gel, sequencing
  • 3T3 - sequencing good > innoculate midi
  • 3T15 - failed gel > mini
  • 6T9 - sequencing not good > mini
  • 9t15 - sequencing not good > mini
  • 12t9 - sequencing not good > mini
  • 12t12 - sequencing not good > mini
  
  
  • 15T3
  • 15T9

07/24

• Midis verified - all correct:
  • 3t9
  • 6t6
  • 6t12
  • 9t3
  • 9t6
  • 12t6
• Failed linkers - mini, digest, gel - all correct > sequencing:
  • 3T15
  • 15T3
  • 15t9
• Correct sequencing

hEF1a:CD79A-XnTCSXc-Gal4VP16