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 DateSummary
07/01
  • hEF1a:CD79B-pTET:mRFP-Gal4VP16 CD79B mutation
  • hEF1a:CD79A-pTET:mRFP-Gal4VP16 Gal4VP16 mutation
  • pLac:mKate miniprep, digest, gel
  • TRE:syk-pTet:mRFP-(t)TEVp miniprep, digest, gel
  • hEF1a:CymR, hEF1a-CuO:mKate, CMV5-CuO:mKate LR
  • pENTR LacI, pENTR hEF1a-LacO transformations
  • TRE:Gal4VP16, TRE:mKate, hEF1a:LacI midi culture

07/02 

Cumate Switch:

  • Kill LR
  • Transform LR product

Lac Switch:

  • digest, gel verify hEF1a-LacO:mKate LR#2
  • midi culture hEF1a-LacO:mKate LR#2 Sample 2

SDM:

MIDIs:

  • TRE:Gal4VP16
  • TRE:mKate
  • hEF1a:LacI

Mini Cultures:

  • pENTR hEF2a-LacO
  • pENTR LacI
07/03
  • Verification digests of all midis + redigests of CD79A linker golden gates
  • midi pLac:mKate
  • mini pENTR hEF1a-LacO and pENTR LacI + digest
  • pick pENTR mKate + mini

SDM plates didn't grow any colonies

07/05
  • Transform TRE:Syk-pTet:RFP-TEV, TRE:Syk-pTet:RFP-tTEV, hEF1a:CD79A-pTet:RFP-gal4VP16 SDM, hEF1a:CD79B-pTet:RFP-Gal4VP16 SDM
  • Plate remainder of failed SDMs
07/06
  • digest, gel:
    • pENTR mKate
    • hEF1a:Gal4VP16 
    • pENTR CD79B-TCS-Gal4VP16
  • sequence:
    • pENTR CD79B-TCS-Gal4VP16
    • TRE:syk-pTET:mRFP-(t)TEVp
  • transform:
    • TRE:syk
    • Re-transform CD79A-TCS-G4 and CD79B-TCS-G4 SDM Golden Gates
  • pick colonies:
    • CD79A-3TCSX-Gal4VP16 colonies
    • CD79A SDM colonies
    • hEF1a:CymR, hEF1a-CuO:mKate, CMV5-CuO:mKate
07/07
  • minprep:
    • CD79A-linker-Gal4VP16 (3T3, 3T9, 3T15)
    • CD79A-pTet:mRFP-Gal4VP16 Gal4 Bsa1 SDM
    • hEF1a:CymR, hEF1a-CuO:mKate, CMV5-CuO:mKate
  • digest, gel:
    • hEF1a:eYFP
    • TRE:eYFP
    • TRE:mKate
    • CD79A-linker-Gal4VP16 (3T3, 3T9, 3T15)
    • CD79A-pTet:mRFP-Gal4VP16 Gal4 Bsa1 SDM
07/08
  • digest hEF1a:CymR, hEF1a-CuO:mKate, CMV5-CuO:mKate
  • TRE:Syk-15-tTEVp PCR
  • Analyze sequencing:
    • TRE:Syk-pTet:mRFP-TEVp - priming issue
    • TRE:Syk-pTet:mRFP-tTEVp - priming issue
    • pENTR CD79B-TCS-G4 - correct
07/09
  • gel:
  • TRE:Syk-15-tTEVp PCR purification
  • TRE:Syk-15-tTEVp golden gate
  • LRs:
    • EGSH:mKate
    • hEf1a:VgEcR-2A-RXR
    • hEF1a:Gal4VP16
    • TRE:mKate
    • hEF1a:CD79B-TCS-Gal4VP16 
    • hEF1a:tagBFP
  • Midi Cultures
07/10
  • Repeat sequencing of CD79A Gal4 SDM #1 since the gal4 read (#4) showed correct mutaiton
  • midi cultures
    • hEF1a:CymR
    • hEF1a-CuO:mKate
    • CMV5-CuO:mKate
    • TRE:Syk
  • Transformations:
    • RE:Syk-15-tTEVp golden gate
    • Golden gate donor
    • LRs:
      • EGSH:mKate
      • hEf1a:VgEcR-2A-RXR
      • TRE:mKate
      • hEF1a:Gal4VP16 (remember which pDEST)
      • hEF1a:CD79B-TCS-Gal4VP16 
      • hEF1a:tagBFP
07/11

 

07/12

 

07/13
  • Reinnoculate midis:
  • Pick colonies:
    • EGSH:mKate
    • hEf1a:VgEcR-2A-RXR
    • TRE:mKate
    • hEF1a:Gal4VP16 (remember which pDEST)
    • hEF1a:CD79B-TCS-Gal4VP16 
    • hEF1a:tagBFP
07/14
  • Midis:
  • Minipreps, digest, gel:
  • cultures didn't grow: hEF1a:gal4, hef1a:VgECr, hef1a: cd79B-TCS-G4, EGSH:mKate --> reinnoculated
  • Golden gates:
    • cd79A-linker-g4
    • TRE:syk-15-ttevp
07/15
  • IgM constant region + RFP PCRs +gel
07/16 
07/17 

 

 

 

Glycerol Stock Map
Spring Semester & May 2015
June 2015

 

 

 

 

 

 

 

DateSummary
03/23Verification of linker-switchable PCRs
04/01
  • rePCRs of CD79B, Gal4 and RFP to make linker switchable constructs
  • Gel verification of 03/31 and 04/01 PCRs
04/10
  • rePCRs of CD79A/B, Gal4 and RFP to make linker switchable constructs
  • Gel verification of PCRs
04/11redesign CD79A/B primers
04/25
  • rePCRs of CD79A/B, to make linker switchable constructs
  • Gel verification of PCRs
04/30
  • rePCR of CD79B with DMSO
  • gel purification of CD79A
  • PCR purification of pTet:mRFP and Gal4VP16
  • AarI golden gate to make hEF1a:CD79A-pTet:mRFP-Gal4VP16
05/01
05/04
05/06
05/29
  • Transformations for Glycerol Stocks
  • Sent for hEF1a:CD79B-TCS-Gal4VP16 sequencing
  • PCRd CD79B
  • CD79B Gel
05/30
  • CD79B Gel Extraction (Failed)
  • Midi Cultures Inocculated
  • CD79B PCR 2
  • Picked Colonies for H:Lyn, H:LT, H:CB-TCS-G4
05/31
  • CD79B PCR Gel
  • CD79B Gel Extraction
  • hEF1a:CD79B-pTet:RFP-Gal4VP16 Golden Gate
  • h:Lyn-TEV colonies picked
DateSummary
06/01
  •  h:Lyn-TEV Colonies Picked
  • h:CDB-RFP-G4 transformation
  • h:CDA-RFP-G4 Sequence Analysis
  • Inocculate Midipreps
  • h:Lyn, h:CDB-TCS-G4 cryostocks
06/02
  • Cloning Redesign
  • Pick hEF1a:CD79B-pTet:RFP-Gal4VP16 Colonies
06/03
  • Miniprep hEF1a:CD79B-pTet:RFP-Gal4VP16
  • Cryostock hEF1a:CD79A-pTet:RFP-Gal4VP16
06/04
  • Glycerol Stock hEF1a:CD79A-pTet:RFP-Gal4VP16
  • Midiprep 4 Frozen pellets
  • Digest & Gel hEF1a:CD79B-pTet:RFP-Gal4VP16
06/08
  • hEF1a:CD79B-pTet:RFP-Gal4VP16 Digest & gel
06/09
  • Send for sequencing
06/11
  • Sequencing Analysis
  • Glycerol Stock H:CDA-RFP-G4
06/16
  • designed SDM primers
  • LR TRE:mKate, hEF1a:LacI, pLac:mKate
  • experimental design + deadlines
06/17
  • annealed linker halves
  • CD79A linker golden gates
06/18
  • transformed CD79A linker golden gates
  • transformed LRs (TRE:mKate, pLac:mKate, hEF1a:LacI)
  • LRed TREt:VP16Gal4
06/19
  • sdm primers are here
  • killed LR TREt:VP16Gal4
  • Transformed LR TREt:VP16Gal4
  • Re-transformed CA-9T6-G4
  • picked CD79A linker colonies
06/21
  • picked CA-9T6-G4 colonies
06/22
  • mini CD79A linkers
  • mini TRE:mKate, pLac:mKate, hEF1a:LacI
  • digest
  • run CD79B SDM gel
06/23
  • digests and gels for TRE:mKate, pLac:mKate, hEF1a:LacI
  • kill TRE:Gal4VP16 LR
  • transform TRE:Gal4VP16 LR
  • run SDM on gel -> gel extraction
  • CD79B-RFP-G4 SDM product transformation
06/24
  • SDM CD79B-RFP-Gal4
  • SDM DpnI digest
  • transform SDM product
  • transform TRE:Gal4VP16
06/25
  • redigest TRE:mKate, pLac:mKate, hEF1a:LacI
  • PCR syk, tevp, ttevp
  • gel verify PCRs and digested LRs
  • TRE:gal4VP16 pick colonies and minipreps
  • PCR purification of syk, tevp, ttevp
  • reLR of TRE:mKate, pLac:mKate, hEF1a:LacI
  • retransform SDM digests
06/26
  • TRE:gal4vp16 gel
  • TRE:syk-mrfp-tevp golden gates
06/28
  • transform golden gates
06/29
  • cultured and miniprepped LR2 TRE:mKate, pLac:mKate, hEF1a:LacI
  • miniprepped LR1 TRE:mKate, pLac:mKate, hEF1a:LacI
  • digest and gel LR1 TRE:mKate, pLac:mKate, hEF1a:LacI
  • picked colonies of new TRE:gal4vp16 transformation

06/30
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