Page History

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 Transform hEF1a:CD79B-pTET:mRFP-Gal4GG Pick pENTR RFP-IgM Colony send CD79A linkers for sequencing
07/22	miniprep, digest, gel pENTR RFP-IgM CD79B, Gal4Mut PCR hEF1a:CD79B-pTET:mRFP-Gal4VP16 golden gate hEF1a:CD79B-pTET:mRFP-Gal4VP16 transformation
07/23	innoculate midis innoculate minis
07/24	midis mini gel digests gel cd79a-linker-gal4vp16: 3t3 3t15 6t9 9t15 12t9 12t12 send for sequencing 3t3 6t9 9t15 12t9 12t12 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

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title	Spring

Date

Summary

Anchor

	5
	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

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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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