Page History

Date	Summary
08/03	miniprep, digest, gel: CD79B-RFP-G4 1,2,3 TRE:Syk-3-tTEVp CD79A-3T15-G4 1,2 CD79A-9T15-G4 1,2 CD79A-15T9-G4 1,2 Midi redigests: A3T3 A12T9 Sequencing A3T15 #1 A9t15 #2 A15T9 #1 CD79B-RFP-G4 #1 TRE:Syk-3-tTEVp
08/04	Midi, digest, gel A-12T12 (good gel - bad yield -> regrew more to midi on 08/05 Miniprep, digest, gel TRE:Syk-6-tTEVp 1,2 Sequencing VRC01L TRE:Syk-6-tTEVp #1 Kill, transform TRE:Syk-15-tTEVp LR Innoculate: A-12T12 midi T:S-3-tT T:S-6-tT T:S-9-tT T:S-12-tT
08/05	Miniprep: T:S-9-tT 1,2 T:S-12-tT 1,2 Midi: A-12T12 Sequence: T:S-9-tT #1 T:S-12-tT #1 A-3T15 #2 A-9T15 #1 Pick: Innoculate:
08/06
08/07	LR TRE:Syk-15-tTEVp (pDEST 2-3) Miniprep, send for sequencing: B-3T3 B-3T9 B-6T6 B-6T9 B-9T3 B-9T6 B-9T12 B-12T6 B-12T9 B-12T12 B-15T3 B-15T15 Miniprep, digest, gel TRE:Syk-15-tTEVp Midiprep, digest, gel TRE:Syk-6-tTEVp Golden Gate hEF1a:CD79A-15T9-G4 (because even though we midi'ed it, sequencing was bad) TRE:Syk-linker-TEVp (3, 6, 9, 12) hEF1a:CD79B-3T15-G4 hEF1a:CD79B-9T9-G4 Pick colonies pENTR HyHEL10 Heavy hEF1a:CD79B-9T15-G4 Restreak hEF1a:CD79B-6T12-G4 hEF1a:CD79B-15T9-G4 (restreak and replate)
08/08	kill LR Transform: LR TRE:Syk-15-tTEVp A-15T9 B-3T15 B-9T9 TRE:Syk-X-TEVp (X=3,6,9,12) Restreak: A-3T15 B-15T15 B-15T9 B-6T12
08/09	Restreak/replate: B9T9 B15T15 Inoculate midis: B3T3 B3T9 B6T6 B9T3 B9T6 B12T6 inoculate minis: TRE:Syk-15-tTEVp -gg TRE:Syk-3-TEVp -gg TRE:Syk-6-TEVp -gg TRE:Syk-9-TEVp -gg TRE:Syk-12-TEVp -gg A15T9 -gg A3T15 A9T15 B3T15 -gg
08/10	Minis: A-3T15 A-9T15 A-15T9 (only one tube) TRE:Syk-3-TEVp TRE:Syk-6-TEVp TRE:Syk-9-TEVp TRE:Syk-12-TEVp B-3T15 B-6T12 B-9T15 TRE:Syk-15-tTEVp --> digest pENTR HyHEL10 H Midis: B3T3 B3T9 B6T6 B9T3 B9T6 B12T6 Golden gate, transform: A3T15 A9T15 Sequencing: A-3T15 1 A-9T15 1 A-15T9 TRE:Syk-3-TEVp 1 TRE:Syk-6-TEVp 1 TRE:Syk-9-TEVp 1 TRE:Syk-12-TEVp 1 B-3T15 1 B-6T12 1 B-9T15 1 TRE:Syk-9-tT 2 TRE: Syk-12-tT 2
08/11
08/12

9/4	Miniprepped and sent 2 copies of B9t9 for sequencing
9/15	LRs (all in pDEST 4-5) hEF1a:Gal4VP16 CuO:VRC01 Heavy CuO:VRC01 Light CuO:HyHEL10 Light CuO:HyHEL10 Heavy
9/16	CuO:VRCO1 Heavy, hEF1a:Gal4VP16, and pENTR Gal4VP16 transformed and plated. CuO: VRCO1 Light, Cuo:HyHEL10 Light and "" Heavy all failed so the failed three were replated using the remainder of the transformation outgrowth culture.
9/17
9/19	TC: HEK293 ft cells (P8) frozen down (at 10^6/mL) - currently in -140C in room 115, back left corner, 1st box in column labelled iGEM cells hEF1a-CuO:VRC01 heavy mini culture in fridge
9/20	Miniprepped : hEF1a-CuO:VRC01 L hEF1a-CuO:HyHEL10 L hEF1a-CuO:HyHEL10 H
9/22	Digest and Gel CuO:VRCOI L CuO:VRCOI H CuO:HyHEL 10 L CuO:HyHEL 10 H All Syk - X - TEV Midis All Syk - X - tTEV Midis hEF1a: Gal4VP16
10/7	Digest CuO:VRC01 L CuO:HyHEL10 L CuO:HyHEL10 H
10/8	Gel CuO:VRC01 L CuO:HyHEL10 L CuO:HyHEL10 H Digest/gel pENTR hEF1a-CuO
10/18	BP and LR Digests
10/20	BP and LR Gels
10/26	LRs (using #1 of pENTRs and pDEST 4-5) CuO:A12T12 CuO:A12T6 CuO:A9T15
11/3	Transform LRs CuO:A12T12 CuO:A12T6 CuO:A9T15
11/4	Pick colonies CuO:A12T12 CuO:A12T6 CuO:A9T15
11/5	Digest CuO:A12T12 CuO:A12T6 CuO:A9T15 Redigest CuO:Gmab H CuO:Gmab L CuO:CD79A CuO:CD79B CuO:Lyn Start midipreps CuO:Gmab H CuO:Gmab L CuO:CD79A CuO:CD79B CuO:Lyn
11/7	Redigest CuO:A12T12 CuO:A12T6 CuO:A9T15 Split cells (P5) - there are three plates (the cells looked really clumpy so I tried different ways of unclumping them, it's possibly because the pipetter wouldn't release properly) Spin down and freeze midiprep cultures
11/15	midi: cuo:gmabh, gmabl, cd79a, cd79b, lyn, cd79a-9t15 mini cuo:cd79a-12t6, cd79a-12t12 more colonies (digest: apa)

Glycerol Stock Map

Spring Semester & May 2015

June 2015

LRs (using #1 of pENTRs and pDEST 4-5)

CuO:A12T12
CuO:A12T6
CuO:A9T15

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title	Spring

Date	Summary
03/23	Verification 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
04/11	redesign 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	Transformation of hEF1a:CD79A-pTET:mRFP-Gal4VP16 golden gate
05/04	hEF1a:CD79A-pTet:mRFP-Gal4VP16 liquid culture
05/06	hEF1a:CD79A-pTet:mRFP-Gal4VP16 miniprep hEF1a:CD79A-pTet:mRFP-Gal4VP16 restriction digest verification
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

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title	June

Date	Summary
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	TRE:gal4vp16 miniprep, digest, gel hef1a:laci, tre:mkate digest, gel (new colonies with old enzymes) LR hef1a:cymR and cmv5CuO:mkate picked colonies for TRE:syk-mrfp-(t)tevp and pLac:mKate

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title	July

Date	Summary
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: PCR purify CD79B QuickChange SDM DpnI digest: hEF1a:CD79B-pTET:mRFP-Gal4VP16 CD79B mutation hEF1a:CD79A-pTET:mRFP-Gal4VP16 Gal4VP16 mutation transform hEF1a:CD79B-pTET:mRFP-Gal4VP16 CD79B mutation hEF1a:CD79A-pTET:mRFP-Gal4VP16 Gal4VP16 mutation 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 sequence: CD79A-pTet:mRFP-Gal4VP16 Gal4 Bsa1 SDM - no priming
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: hEF1a:CymR, hEF1a-CuO:mKate, CMV5-CuO:mKate TRE:Syk-15-tTEVp PCR 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 hEF1a:CymR, hEF1a-CuO:mKate TRE:Syk
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: hEF1a:CymR hEF1a-CuO:mKate CMV5-CuO:mKate TRE:Syk Pick colonies: EGSH:mKate hEf1a:VgEcR-2A-RXR TRE:mKate hEF1a:Gal4VP16 (remember which pDEST) hEF1a:CD79B-TCS-Gal4VP16 hEF1a:tagBFP
07/14	Midis: hEF1a:CymR hEF1a-CuO:mKate CMV5-CuO:mKate TRE:Syk Minipreps, digest, gel: TRE:mKate hEF1a:tagBFP hEF1a:CymR hEF1a-CuO:mKate 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 IgM-RFP golden gate Transform CD79A linker golden gates and remainder of LRs for cultures that didn't grow today: TRE:Syk-15-tTEVp TRE:mKate EGSH:mKate hEF1a:VgEcR-2A-RxR hEF1a:Gal4VP16 hEF1a:CD79B-TCS-Gal4VP16 Ordered Primers: TRE:Syk-pTet:mRFP-TEVp sequencing primers hEF1a:Cd79B-pTet:mRFP-Gal4VP16 PCR primers
07/16	innoculate: TRE:Syk-15-tTEVp TRE:mKate EGSH:mKate hEF1a:VgEcR-2A-RxR hEF1a:Gal4VP16 hEF1a:CD79B-TCS-Gal4VP16 CD79A-3T3-Gal4 CD79A-6T6-G4 CD79A-6T12-G4 CD79A-9T3-G4 replate CD79A linker golden gate transformations that didn't give colonies
07/17	replated golden gates didn't give any colonies (may have used too little DNA) miniprep, digest, gel: TRE:Syk-15-tTEVp TRE:mKate EGSH:mKate hEF1a:VgEcR-2A-RxR hEF1a:Gal4VP16 hEF1a:CD79B-TCS-Gal4VP16 CD79A-3T3-Gal4 CD79A-6T6-G4 CD79A-6T12-G4 CD79A-9T3-G4 CD79A linker golden gates innoculate midis
07/18	CD79A linker golden gate T4 ligation Transform CD79A golden gates Midipreps: hEF1a:Gal4 hEF1a:tagBFP hEF1a:VgEcR-2A-RxR EGSH:mKate hEF1a:CD79B-TCS-Gal4VP16 TRE:mKate
07/19	CD79A linker golden gates liquid cultures
07/20	Miniprep CD79A golden gate liquid cultures Digest, gel: CD79A linker hEF1a:Gal4 hEF1a:tagBFP hEF1a:VgEcR-2A-RxR EGSH:mKate hEF1a:CD79B-TCS-Gal4VP16 TRE:mKate IgM-RFP pENTR golden gate transformation
07/21	CD79B & Gal4VP16 PCRs Gel to test Oligo Annealing Anneal Oligos in annealing buffer Golden Gates hEF1a:CD79B-pTET:mRFP-Gal4 GG TRE:Syk-15-tTEVp GG Transform hEF1a:CD79B-pTET:mRFP-Gal4GG TRE:Syk-15-tTEVp GG Pick pENTR RFP-IgM Colony Sequencing: CD79A linkers TRE:Syk-pTet:mRFP-tTEVp remaining read
07/22	miniprep, digest, gel pENTR RFP-IgM CD79B, Gal4Mut PCR hEF1a:CD79B-pTET:mRFP-Gal4VP16 golden gate (failed) hEF1a:CD79B-pTET:mRFP-Gal4VP16 transformation
07/23	innoculate midis of correct CD79A linker constructs: 3t9 6t6 6t12 9t3 9t6 12t6 innoculate minis: hEF1a:gal4vp16 3t15 6T9 9T12 9T15 12T9 12T12 15t9 analyze TRE:syk-pTet:mRFP-tTEVp sequencing (no priming) resend TRE:syk-pTet:mRFP-tTEVp for sequencing
07/24	gel cd79a-linker-gal4vp16: 3t3 3t15 6t9 9t15 12t9 12t12 send for sequencing 3t3 6t9 9t15 12t9 12t12 midis 3t9 6t6 6t12 9t3 9t6 12t6 mini digests gels 3t15 15t9 hEF1a:gal4 rePCR RFP Golden gates hEF1a:CD79B-pTET:mRFP-Gal4 syk linker tTEVp golden gates Transformations hEF1a:CD79B-pTET:mRFP-Gal4 syk linker ttevp golden gates mini, digest, gel 6T9 9T12 9T15 12T9 12T12 send for sequencing: 3T15 15T9
07/25
07/26
07/27	AarI verification gel CD79A linker midis innoculate CD79A linker midis innoculate Syk-linker minis innoculate CD79A failed linker minis Golden gates: VRC01 heavy + light pENTR HyHel10 heavy + light pENTR
07/28	Mini, digest, gel: TRE:Syk-3-tTEVp TRE:Syk-6-tTEVp TRE:Syk-9-tTEVp TRE:Syk-12-tTEVp hEF1a:CD79A-9T15-G4 Midi, digest, gel: CD79A-6T9-G4 CD79A-9T9-G4 CD79A-9T12-G4 CD79A-15T3-G4 CD79A-15T15-G4 hEF1a:Gal4 Sequencing: resend 12T12 9T15 Transform Golden gates: VRC01 heavy + light pENTR HyHel10 heavy + light pENTR Syk-15-tTEVp retransformation CD79B-RFP-G4 retransformation
07/29	Mini, digest gel: CD79A-3T15-G4 CD79A-12T9-G4 CD79A-15T9-G4 Golden Gates Sequencing
07/30	mini, digest, gel midi, digest, gel A-3T3 GG Donr Innoculate Mini cultures with successful GGs Innoculate midi culture Replated failed GG transformations TRE:Syk-6-tTEVp TRE:Syk-9-tTEVp sequencing: 3T15 - okay -> resequencing 15T9 - bad VRC01 H pENTR - good VRC01 L pENTR - bad quality -> resequencing HyHEL10 H pENTR - bad quality -> resequencing
07/31	mini, digest, gel CD79A-12T12-G4 Syk-15-tTEVp pENTR HyHel10 Light pENTR Midi, digest, gel A-12T9 Innoculate minis TRE:Syk-12-tTEVp TRE:Syk-3-tTEVp hEF1a:CD79B-pTET:mRFP-G4 A-15T9 A-12T12 A-9T15 A-3T15 Sequencing A-12T12 HyHEL 10 L pENTR Syk-15-tTEVp pENTR

Summary

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

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title	August

Date	Summary
08/03	miniprep, digest, gel: CD79B-RFP-G4 1,2,3 TRE:Syk-3-tTEVp CD79A-3T15-G4 1,2 CD79A-9T15-G4 1,2 CD79A-15T9-G4 1,2 Midi redigests: A3T3 A12T9 Sequencing A3T15 #1 A9t15 #2 A15T9 #1 CD79B-RFP-G4 #1 TRE:Syk-3-tTEVp
08/04	Midi, digest, gel A-12T12 (good gel - bad yield -> regrew more to midi on 08/05 Miniprep, digest, gel TRE:Syk-6-tTEVp 1,2 Sequencing VRC01L TRE:Syk-6-tTEVp #1 Kill, transform TRE:Syk-15-tTEVp LR Innoculate: A-12T12 midi T:S-3-tT T:S-6-tT T:S-9-tT T:S-12-tT
08/05	Miniprep: T:S-9-tT 1,2 T:S-12-tT 1,2 Midi: A-12T12 Sequence: T:S-9-tT #1 T:S-12-tT #1 A-3T15 #2 A-9T15 #1 Pick: Innoculate:
08/06
08/07	LR TRE:Syk-15-tTEVp (pDEST 2-3) Miniprep, send for sequencing: B-3T3 B-3T9 B-6T6 B-6T9 B-9T3 B-9T6 B-9T12 B-12T6 B-12T9 B-12T12 B-15T3 B-15T15 Miniprep, digest, gel TRE:Syk-15-tTEVp Midiprep, digest, gel TRE:Syk-6-tTEVp Golden Gate hEF1a:CD79A-15T9-G4 (because even though we midi'ed it, sequencing was bad) TRE:Syk-linker-TEVp (3, 6, 9, 12) hEF1a:CD79B-3T15-G4 hEF1a:CD79B-9T9-G4 Pick colonies pENTR HyHEL10 Heavy hEF1a:CD79B-9T15-G4 Restreak hEF1a:CD79B-6T12-G4 hEF1a:CD79B-15T9-G4 (restreak and replate)
08/08	kill LR Transform: LR TRE:Syk-15-tTEVp A-15T9 B-3T15 B-9T9 TRE:Syk-X-TEVp (X=3,6,9,12) Restreak: A-3T15 B-15T15 B-15T9 B-6T12
08/09	Restreak/replate: B9T9 B15T15 Inoculate midis: B3T3 B3T9 B6T6 B9T3 B9T6 B12T6 inoculate minis: TRE:Syk-15-tTEVp -gg TRE:Syk-3-TEVp -gg TRE:Syk-6-TEVp -gg TRE:Syk-9-TEVp -gg TRE:Syk-12-TEVp -gg A15T9 -gg A3T15 A9T15 B3T15 -gg
08/10	Minis: A-3T15 A-9T15 A-15T9 (only one tube) TRE:Syk-3-TEVp TRE:Syk-6-TEVp TRE:Syk-9-TEVp TRE:Syk-12-TEVp B-3T15 B-6T12 B-9T15 TRE:Syk-15-tTEVp --> digest pENTR HyHEL10 H Midis: B3T3 B3T9 B6T6 B9T3 B9T6 B12T6 Golden gate, transform: A3T15 A9T15 Sequencing: A-3T15 1 A-9T15 1 A-15T9 TRE:Syk-3-TEVp 1 TRE:Syk-6-TEVp 1 TRE:Syk-9-TEVp 1 TRE:Syk-12-TEVp 1 B-3T15 1 B-6T12 1 B-9T15 1 TRE:Syk-9-tT 2 TRE: Syk-12-tT 2
08/11	Run gel of TRE:syk-15-tTEVp: correct, inoculate midi digest pENTR HyHEL10 H Sequencing: B6T9-1 B9T12-1 Inoculate midi: B15T3 T:S-15-tT Inoculate mini: B9T9 - from plate A3T15 - from plate Restreak/replate: B15T9 A9T15 - one white B15T15 B9T9 - only one white
08/12	Run gel for pENTR HyHEL10 H digest Transform: B15T15 Mini, sequence: A9T15 B9T9 Midi: TRE:syk-15-tTp inoculate midi: B15T3 T:S-9-TEVp T:S-12-TEVp B9T12 inoculate mini: A9T15 B15T9 B6T9 Sequencing B6T12-2 pENTR HyHEL10H-1 A15T9-2 B9T9 (from 8/12) Repeat sequencing: T:S-3-T 1 T:S-6-T 1 T:S-9-tT 2 T:S-12-tT 2 B3T15 1 B9T15 1 B12T12 2
08/13	Midis: B9T12 B15T3 S9TEV S12TEV Minis, sequencing B6T9 A9T15 Inoculate mini B15T9 B15T15 B12T9 T:S-9-tT T:S-12-tT Inoculate midi: B12T12-2 Sequencing: B6T9-1 A9T15-2 A3T15-2 T:S-3-T 1 T:S-6-T-1
08/14	Midis B12T12-2 Minis, sequencing T:S-9-tT T:S-12-tT Order antibodies/antigens Finish Gaussian Distribution gating code Split Cells Inoculate mini B15T15 B6T12 B6T9 A3T15 Hef1a:GAL4? Retransform B9T9 A15T9 T:S-6-T B15T9 B12T9 Sequencing, take 1 B9T15-2 B3T15-1 T:S9tT 1 T:S12tT 1 Sequencing, take 2 T:S3T #2 A9T15 #1 (8/12)
08/15	Miniprep/sequence B15T15 - one red, one with no growth :/ B6T12 B6T9 A3T15 Inoculate minis B9T9 A15T9 T:S-3-T T:S-6-T B12T9 B15T15 Analyze sequencing and go from there
08/16	Make amp plates Replate B12T9 B15T9 Inoculate miniprep cultures A15T9 T:S-6-T B9T9 Inoculate midiprep culture A9T15 B6T9 probably B6T12 B6T12 probably B6T9 Miniprep and sequence T:S-3-T B15T15 Spin down and store midiprep cultures T:S-9-tT T:S-12-tT Analyze sequencing data B6T12 B6T9 A3T15
08/17	Send for sequencing A3T15 #2 (8/15) B3T15 #1 B9T15 #1 Replate B12T9 B15T9 Inoculate miniprep cultures A15T9 T:S-6-T B9T9 Inoculate midiprep culture T:S-3-TEV Spin down and store midiprep cultures
08/18	Golden gate, transform A15T9 B3T15 B9T9 B9T15 B12T9 B15T9 Transform lab stock of Gal4VP16 Miniprep, sequence T:S-6-T Inoculate miniprep A3T15 A15T9 Spin down midiprep T:S-3-TEV
08/19
08/20

Expand

title	Spring

Date

Anchor

5

03/23

Verification 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

04/11

redesign 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

Transformation of hEF1a:CD79A-pTET:mRFP-Gal4VP16 golden gate

05/04

hEF1a:CD79A-pTet:mRFP-Gal4VP16 liquid culture

05/06

hEF1a:CD79A-pTet:mRFP-Gal4VP16 miniprep
hEF1a:CD79A-pTet:mRFP-Gal4VP16 restriction digest verification

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

Expand

title	June

Date

Summary

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

06/30

TRE:gal4vp16 miniprep, digest, gel
hef1a:laci, tre:mkate digest, gel (new colonies with old enzymes)
LR hef1a:cymR and cmv5CuO:mkate
picked colonies for TRE:syk-mrfp-(t)tevp and pLac:mKate

Expand

title	July

mini, digest, gel

CD79A-12T12-G4
Syk-15-tTEVp pENTR
HyHel10 Light pENTR

Midi, digest, gel

A-12T9

Innoculate minis

TRE:Syk-12-tTEVp
TRE:Syk-3-tTEVp
hEF1a:CD79B-pTET:mRFP-G4
A-15T9
A-12T12
A-9T15
A-3T15

Sequencing

A-12T12

HyHEL 10 L pENTR

Syk-15-tTEVp pENTR

Date	Summary
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: PCR purify CD79B QuickChange SDM DpnI digest: hEF1a:CD79B-pTET:mRFP-Gal4VP16 CD79B mutation hEF1a:CD79A-pTET:mRFP-Gal4VP16 Gal4VP16 mutation transform hEF1a:CD79B-pTET:mRFP-Gal4VP16 CD79B mutation hEF1a:CD79A-pTET:mRFP-Gal4VP16 Gal4VP16 mutation 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 sequence: CD79A-pTet:mRFP-Gal4VP16 Gal4 Bsa1 SDM - no priming
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: hEF1a:CymR, hEF1a-CuO:mKate, CMV5-CuO:mKate TRE:Syk-15-tTEVp PCR 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 hEF1a:CymR, hEF1a-CuO:mKate TRE:Syk
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: hEF1a:CymR hEF1a-CuO:mKate CMV5-CuO:mKate TRE:Syk Pick colonies: EGSH:mKate hEf1a:VgEcR-2A-RXR TRE:mKate hEF1a:Gal4VP16 (remember which pDEST) hEF1a:CD79B-TCS-Gal4VP16 hEF1a:tagBFP
07/14	Midis: hEF1a:CymR hEF1a-CuO:mKate CMV5-CuO:mKate TRE:Syk Minipreps, digest, gel: TRE:mKate hEF1a:tagBFP hEF1a:CymR hEF1a-CuO:mKate 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 IgM-RFP golden gate Transform CD79A linker golden gates and remainder of LRs for cultures that didn't grow today: TRE:Syk-15-tTEVp TRE:mKate EGSH:mKate hEF1a:VgEcR-2A-RxR hEF1a:Gal4VP16 hEF1a:CD79B-TCS-Gal4VP16 Ordered Primers: TRE:Syk-pTet:mRFP-TEVp sequencing primers hEF1a:Cd79B-pTet:mRFP-Gal4VP16 PCR primers
07/16	innoculate: TRE:Syk-15-tTEVp TRE:mKate EGSH:mKate hEF1a:VgEcR-2A-RxR hEF1a:Gal4VP16 hEF1a:CD79B-TCS-Gal4VP16 CD79A-3T3-Gal4 CD79A-6T6-G4 CD79A-6T12-G4 CD79A-9T3-G4 replate CD79A linker golden gate transformations that didn't give colonies
07/17	replated golden gates didn't give any colonies (may have used too little DNA) miniprep, digest, gel: TRE:Syk-15-tTEVp TRE:mKate EGSH:mKate hEF1a:VgEcR-2A-RxR hEF1a:Gal4VP16 hEF1a:CD79B-TCS-Gal4VP16 CD79A-3T3-Gal4 CD79A-6T6-G4 CD79A-6T12-G4 CD79A-9T3-G4 CD79A linker golden gates innoculate midis
07/18	CD79A linker golden gate T4 ligation Transform CD79A golden gates Midipreps: hEF1a:Gal4 hEF1a:tagBFP hEF1a:VgEcR-2A-RxR EGSH:mKate hEF1a:CD79B-TCS-Gal4VP16 TRE:mKate
07/19	CD79A linker golden gates liquid cultures
07/20	Miniprep CD79A golden gate liquid cultures Digest, gel: CD79A linker hEF1a:Gal4 hEF1a:tagBFP hEF1a:VgEcR-2A-RxR EGSH:mKate hEF1a:CD79B-TCS-Gal4VP16 TRE:mKate IgM-RFP pENTR golden gate transformation
07/21	CD79B & Gal4VP16 PCRs Gel to test Oligo Annealing Anneal Oligos in annealing buffer Golden Gates hEF1a:CD79B-pTET:mRFP-Gal4 GG TRE:Syk-15-tTEVp GG Transform hEF1a:CD79B-pTET:mRFP-Gal4GG TRE:Syk-15-tTEVp GG Pick pENTR RFP-IgM Colony Sequencing: CD79A linkers TRE:Syk-pTet:mRFP-tTEVp remaining read
07/22	miniprep, digest, gel pENTR RFP-IgM CD79B, Gal4Mut PCR hEF1a:CD79B-pTET:mRFP-Gal4VP16 golden gate (failed) hEF1a:CD79B-pTET:mRFP-Gal4VP16 transformation
07/23	innoculate midis of correct CD79A linker constructs: 3t9 6t6 6t12 9t3 9t6 12t6 innoculate minis: hEF1a:gal4vp16 3t15 6T9 9T12 9T15 12T9 12T12 15t9 analyze TRE:syk-pTet:mRFP-tTEVp sequencing (no priming) resend TRE:syk-pTet:mRFP-tTEVp for sequencing
07/24	gel cd79a-linker-gal4vp16: 3t3 3t15 6t9 9t15 12t9 12t12 send for sequencing 3t3 6t9 9t15 12t9 12t12 midis 3t9 6t6 6t12 9t3 9t6 12t6 mini digests gels 3t15 15t9 hEF1a:gal4 rePCR RFP Golden gates hEF1a:CD79B-pTET:mRFP-Gal4 syk linker tTEVp golden gates Transformations hEF1a:CD79B-pTET:mRFP-Gal4 syk linker ttevp golden gates mini, digest, gel 6T9 9T12 9T15 12T9 12T12 send for sequencing: 3T15 15T9
07/25
07/26
07/27	AarI verification gel CD79A linker midis innoculate CD79A linker midis innoculate Syk-linker minis innoculate CD79A failed linker minis Golden gates: VRC01 heavy + light pENTR HyHel10 heavy + light pENTR
07/28	Mini, digest, gel: TRE:Syk-3-tTEVp TRE:Syk-6-tTEVp TRE:Syk-9-tTEVp TRE:Syk-12-tTEVp hEF1a:CD79A-9T15-G4 Midi, digest, gel: CD79A-6T9-G4 CD79A-9T9-G4 CD79A-9T12-G4 CD79A-15T3-G4 CD79A-15T15-G4 hEF1a:Gal4 Sequencing: resend 12T12 9T15 Transform Golden gates: VRC01 heavy + light pENTR HyHel10 heavy + light pENTR Syk-15-tTEVp retransformation CD79B-RFP-G4 retransformation
07/29	Mini, digest gel: CD79A-3T15-G4 CD79A-12T9-G4 CD79A-15T9-G4 Golden Gates Sequencing
07/30	mini, digest, gel midi, digest, gel A-3T3 GG Donr Innoculate Mini cultures with successful GGs Innoculate midi culture Replated failed GG transformations TRE:Syk-6-tTEVp TRE:Syk-9-tTEVp sequencing: 3T15 - okay -> resequencing 15T9 - bad VRC01 H pENTR - good VRC01 L pENTR - bad quality -> resequencing HyHEL10 H pENTR - bad quality -> resequencing
07/31

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