Notebook Overview

Our team has been working hard on our project for many months, and we are proud to share our exciting results with the iGEM community! Here we present a summary of our daily progress and experiments. Starting in the 2013 spring semester, our team learned laboratory techniques and began experiments. Over the summer, the full 10 member team - including one undergraduate student from another university - worked full-time on the project. Currently, during the 2013 fall semester, some members of the team are continuing to invest time and conduct experiments for the project through the Undergraduate Research Opportunities Program at MIT.

Construction and In Vitro Lab Notebook

Date	Team Member	Experiments
5/8/2013	KL	LR reaction to make hEF1a_mKate, hEF1a _TagBFP, hEF1a_rtTA3, TRE-t_mKate, hEF1a_eYFP, hEF1a_eBFP2
5/10/2013	KL	Transformation with LR reaction results
5/13/2013	KL	Miniprep (DM 6 yeast constructs) and Restriction Digests (DM 6 yeast constructs)
5/14/2013	KL	Gels of digested 6 yeast constructs
5/14/2013	NH	"Inoculation of iGEM 2012 stocks for transfection by Nelson: hEF1a_rtTA3 to test TET System,
hEF1a_mKate for transfection controls, hEF1a_eYFP for transfection controls, hEF1a_eBFP2 for transfection controls, hEF1a_TagBFP for transfection controls, TRE-tight_mKate to test TET System, TRE-tight_eYFP to test TET System, pGG_DONR for building GG ENTRs of TyA-eGFP
"
5/15/2013	KL	Miniprep of LR2 plasmids, digestion of LR2 plasmids with EcoRI, and transformation of ENTR stock plasmids for propagation: eBFP, eYFP, rtTA3, mKATE, TagBFP, CAG, hEF1a, TRE-t, CMV
5/16/2013	KL	Gel of LR2 plasmids, New LR reaction to produce 1-2_TRE-T_mKate and inoculation of liquid cultures of ENTR stock plasmids
5/17/2013	KL	Miniprep of ENTR stock plasmids, sent for sequencing, preparation of glycerol stock cultures of ENTR plasmids, and transformation of 1-2_TRE-t_mKate produced by LR reaction.
5/21/2013	KL	Miniprep of DM cultures, inoculation of 12 liquid cultures + amp with colonies produced after transformation with 1-2_TRE-t_mKate LR product, and reviewed sequences of ENTR plasmids
5/22/2013	KL	24 Minipreps - DM cultures, reviewed sequences of three ENTRs that were resubmitted, accepted all three, 8 of the 9 ENTRs have had their sequence verified, missing only CAG, inoculated 6 liquid cultures with ENTR_CAG for miniprep and resequencing, prepared 5fmol stocks of ENTRs, and made LR reactions of 1-2_CMV_eYFP, 1-2_CMV_mKate, 2-3_CMV_eYFP, 2-3_CMV_mKate
5/23/2013	KL	Miniprep of 6 liquid cultures of ENTR_CAG
5/24/2013	KL	Transformation and plating with products of LR reactions: 1-2_CMV_eYFP, 1-2_CMV_mKate, 2-3_CMV_eYFP, 2-3_CMV_mKate
5/28/2013	KL	Design and ordering of DNA for Acyl-TyA-GFP experiment
5/29/2013	KL	LR reactions to make TRE-t_mKate, CMV_mKate, CMV_eBFP2, hEF1a_eYFP, miniprep of 3 DM cultures, and prepared and sent for sequencing 6 ENTR_CAG plasmids miniprepped on 5/23
5/30/2013	KL	transformation of LR Reactions: TRE-t_mKate, CMV_mKate, CMV_eBFP2, hEF1a_eYFP, started design of eYFP-tsmiR451 and eYFP-tsmiR503 and intronic miR451, inoculation of 50mL flasks for midiprep: 1-2_hEF1a_eBFP2, 1-2_hEF1a_rtTA3, 1-2_hEF1a_mKate,1-2_TRE-t_eYFP
5/31/2013	KL	"Midiprepped 50mL cultures: 1-2_hEF1a_eBFP2, 1-2_hEF1a_rtTA3, 1-2_hEF1a_mKate,1-2_TRE-t_eYFP, mixed up resulting DNA, so doing restriction maps to determine which is which.
Enzymes:
BamHI will distinguish TRE-T: eYFP from the other three
ApaLI will distinguish Hef1A: mKate
SalI will distinguish Hef1A: eBFP2
Which will leave Hef1A: rtTA3."
6/3/2013	KL	Design of eYFP-4xmiR451a and miR503
6/4/2013	KL	Sent midis for sequencing: Mystery1-sGIB001, Mystery2-sGIB001, Mystery3-sGIB001, Mystery4-sGIB001, Mystery1-sGIB003, Mystery2-sGIB003, Mystery4-sGIB003, Mystery3-sGIB003, inoculated 1-2, 2-3, 3-4 DEST vectors for minipreps (AMP, CM selection – 5mL SOB cultures), grow at 30C, and inoculated 10G cells for competent cells (one aliquot into 5mL TB, super sterile)
6/5/2013	KL	Prepared stocks for iGEM: 3x 900mL LB, 4x 500mL LB-A, 2x 900mL SOB, 1x 900mL sH20, 10x 1mL of AMP, KAN, CM, 18x 50mL of SOC; From sequencing, determined Mystery Midis: 1 - hEF1a_rtTA3, 2 - hEF1a_mKate, 3 - hEF1a_eBFP2, 4 - TRE-t_eYFP, and made competent cells
6/5/2013	KL/NH	Designed sequencing primers for ENTR vectors matching the gene/promoter so that we can sequence the EXPR plasmids from iGEM 2012.
6/6/2013	KL	Miniprep of DEST cultures from Jameel
6/13/2013	CL	Prepared Miniprep and Midiprep cultures of transformed E. Coli: VN173 , VC155, VN-V150A
6/14/2013	NH	"Transform competent bacteria with TRE-mkate and hEF1a_-rtTA, transform competent bacteria
with TRE-GAL4/VP16, design and order the VP16 gBlock"
6/14/2013	CL	Purified plasmids with Midiprep kit. Nanodropped and stored samples @-20, striked AMP plates with each construct and allowed cells to grow overnight @37: Traffic Light Reporter and px335-U6 Chimeric BB - cBh hsCAS9
6/15/2013	CL	Picked colony and prepared 50ml Midiprep culture
6/16/2013	CL	Midiprep for constructs
6/17/2013	NH	Pick a colony from TRE-GAL4/VP16 and culture the bacteria for midiprepping
6/18/2013	KL	Golden gate: gBlock 001: Q1-Kozak-ATG-EYFP-Q2, gBlock 002: Q2-EYFP-4xmiR541-Qx, gBlock 003: Q2-EYFP-4xmiR509-Qx with pDONR: L1-Q1-LacZ-Qx-L2-P15A-KanR to make L1-Q1-Kozak-ATG-EYFP-4xmiR451(509)-Qx-L2-P15A-KanR
6/18/2013	NH	Re-do the Gal4VP16 LR reaction, Run a gel of the Gal4VP16 LR, Midiprep the plasmids made yesterday, Make Kan+ and Amp+ plates
6/19/2013	KL	Transform golden gate L1-Q1-Kozak-ATG-EYFP-4xmiR451(509)-Qx-L2-P15A-KanR
6/19/2013	NH	Sequence everything from 6/18-NH, Make even more LB-A, Make Kan+ and Amp+ plates, Design the stockroom page
6/20/2013	KL	"Pick colonies from 6/19-KL, Picked 5 white colonies from each GG plate (eYFP-4xmiR451 and eYFP-4xmiR503) and inoculated each in 4mL LB+Kan
37C shaker overnight"
6/21/2013	NH	Practice transformation with Hyo
6/21/2013	KL	Standard miniprep of 6/20-KL, nanodropped, digestion of miniprep with BsrGI, sent miniprep samples for sequencing
6/24/2013	NH	Check the sequences of the Gal4-VP16 and then re-LR it, Order Cas9-VP16 string, practice transformation/digestions/LR's with Hyo
6/25/2013	NH	Practice Midi-prep with Hyo & Molly
6/25/2013	BN	Transformed ENTR_L1_D10A & H840A mutated cas9_C-terminal linker_L2
6/26/2013	KL	Dilution to 5fmol stock of minis from 6/21-KL, LR reaction to make EXPR_23_Hef1A_eYFP-4xmiR451, EXPR_23_Hef1A_eYFP-4xmiR503, EXPR_23_TRE-T_eYFP-4xmiR451, EXPR_23_TRE-T_YFP-4xmiR503
6/26/2013	MK	TRE-tight-GAL4VP16 LR rxn
6/26/2013	MP	PCR ENTR_L1_HA-mKate_L2
6/27/2013	NH	Set up Hef1A - GAL4VP15 LR rxn, Get and send for sequencing the 5x UAS miniCMV entry vector, Transform LR last time.
6/27/2013	KL	Transformation of LR Products from 6/26-KL
6/27/2013	BN	"inoculated ENTR_L1_D10A & H840A mutated cas9_C-terminal linker_L2
"
6/27/2013	LS	Gel Extraction + BP + Transformation
6/28/2013	KL	Pick colonies of LR Products, Inoculated 4mL cultures of LB with Amp
6/28/2013	NH	Get the Cas9-linker-BsaI site entry vector sequenced, Label the tubes
6/28/2013	HX	Inoculate cells
6/29/2013	KL	Pick colonies of LR Products again, Inoculated 4mL cultures of LB with Amp
6/29/2013	KL	PCR to amplify mKate Exons and Ran a gel to purify the PCR product
6/30/2013	KL	Miniprep culture from 6/29-KL, Restriction Map, Re-do LR of eYFP-miR503s
7/1/2013	KL	Transform eYFP-miR503s, send Miniprep from 6/30-KL for sequencing
7/1/2013	KL	Re-do PCR to amplify mKate Exon 2
7/2/2013	KL	Pick eYFP-miR503s colonies, Inoculate 4mL cultures, 1x mKate Golden Gates to make L1-Q1-mKate Exon 1-1x miR451-mKate Exon 2-Qx-L2-P15A-KanR
7/2/2013	NH	"Digestion verify Hef1A-Gal4Vp16 and TRE-tight-Gal4Vp16
"
7/2/2013	MP	Miniprep ENTR_L1_HA-mKate_L2
7/2/2013	KL	Ran Gel extraction of 7/1-KL on a 1% agarose gel, performed Qiagen gel extraction protocol
7/3/2013	NH	Preformed digestion on Hef1A-Gal4Vp16 and TRE-tight-Gal4Vp16, U6-gRNA, EXPR-EYFP
7/3/2013	CL	Goldengate Cas9-VC155, Cas9-VN155, Cas9-VN173, VN155-Cas9, VN173-Cas9, VC155-Cas9, BsaI digest and ligation nd restriction mapped
7/3/2013	KL	Miniprep from 7/2-KL culture, Transform 1x mKate from 7/2-KL, PCR to amplify Exon 1, Exon 1 Gel Extraction, Re-do 1x mKate Golden Gates
7/4/2013	KL	Transform 1x mKates from 7/3-KL Goldengates
7/4/2013	MP	Digest and restriction map, ENTR_L1_HA-mKate_L2
7/4/2013	CL	Transformed Golden gates from 7/3-CL
7/4/2013	NH	Preformed a gel on the digestions to verify, sent them out for sequencing
7/5/2013	KL	Restriction Map 7/3-KL miniprep
7/5/2013	HX	Miniprep ENTR_L1_HA-mKate_L2 and send for sequencing
7/5/2013	KL	Pick 1x mKate colonies from 7/4-KL transform
7/5/2013	NH	Transformed bacteria with DNA from the four constructs, plated
7/6/2013	NH	Picked colonies from 7/5-NH
7/6/2013	KL	Miniprep 1x mKates and restriction map
7/7/2013	KL	Redo LR of (Hef1A/CAG)_eYFP-miR503 and LR Hef1A and TRE-t with 1x mKates
7/7/2013	NH	Mini prepped the Hef1A-Gal4Vp16, TRE-tight-Gal4Vp16, U6-gRNA, EXPR-EYFP and Prepared midi culture with Hef1A-Gal4Vp16 and TRE-tight-Gal4Vp16
7/8/2013	KL	Transform Hef1A and TRE-t with 1x mKates: 23_Hef1A_mKate-1xmiR451, 23_Hef1A_mKate-1xmiR503, 23_TRE-t_mKate-1xmiR451, 23_TRE-t_mKate-1xmiR503, transform (Hef1A/CAG)_eYFP-miR503,
7/8/2013	CL	Miniprepped three cultures of LR products of CAG_split venus with or without leucine zippers except CAG_VN173 w/ zip because the plate had a lawn and the outgrowth had to be replated to be able to pick a single colony for inoculation
7/8/2013	MK	Transformed pDONR P4P1r to 10G competent cells and midiprepped
7/8/2013	NH	Transformed DONR_P4P1r, VP16_N-Term, VP16_C-Term.
7/9/2013	KL	Picked new colonies of mKate-1xmiR451
7/9/2013	CL	Restriction mapping of Cas9-splitvenusproteins
7/9/2013	NH	Midi prep Hef1A-Gal4Vp16 and TRE-tight-Gal4Vp16, Plan Goldengate RXN with VP16 gBlocks and Cas9-Linker constructs. (concentrations, protocol, getting the DNA in one location ready to go.), Make LB-Agar, Make Plates
7/10/2013	KL	Picked colonies of Hef1A_eYFP-miR503
7/10/2013	CL	Restriction map of C-terminal Cas9 fusion proteins (cut with PvuII)
7/10/2013	KL	Miniprep ENTR_mKate-1xmiR451 and restriction map, Picked colonies of TRE-t_mKate-1xmiR503, do LR of EXPR_mKates
7/10/2013	NH	Check up on GAL4VP16 experiments. If okay, then go ahead and transfect the TRE-tight GAL4VP16's, Midi prep Hef1A-Gal4Vp16 and TRE-tight-Gal4Vp16, Prepare midi culture with DONR_P4P1r, VP16_N-Term, VP16_C-Term, Plan Goldengates! then do them and plate them
7/11/2013	KL	Miniprep Hef1A_eYFP-miR503 and restriction map
7/11/2013	NH	Miniprep goldengate product and Midiprep pDONR_P4P1r, VP16_N-Term, VP16_C-Term
7/11/2013	KL	Restriction map of ENTR_mKate-1xmiR451 and Transform EXPR_mKates
7/12/2013	CL	PCR cas9 with attB1 and attB2 sites
7/12/2013	KL	Picked colonies of EXPR_mKates and more of ENTR_mKate-miR45
7/13/2013	KL	Miniprep EXPR_mKates and ENTR_mKate-miR451 and restriction mapped, Midiprep Hef1A_eYFP-miR503
7/13/2013	CL	Restriction map of VC155-Cas9 and VN155-Cas9 with PciI and PvuI
7/14/2013	KL	Midiprep of EXPR_mKates: Hef1A_mKate-1xmiR503, Hef1A_mKate-1xmiR451, TRE-T_mKate-1xmiR451
7/15/2013	CL	VN173-Cas9 restriction map with PvuI and PciI
7/16/2013	NH	"Miniprep all samples, Run a diagnostic gel on the BP product and topo vector, Transform P4P1r donor in ccdB resistant bacteria, Send all samples for sequencing
"
7/16/2013	MK	Re-transformation
7/16/2013	CL	Restriction map with BamHI and SalI
7/17/2013	KL	LR of EXPR_mKate-451s: Hef1A_mKate-miR451 and TRE-t_mKate-miR451
7/17/2013	NH	Purify out PCR product, Re-Goldengate VP16-Cas9, Assess the Digestions and determine whether or not we should send for sequencing, Send BP product to sequencing (use m13 primers) and the P4P1r donor, Figure out Topovector gel situation - make sure that the enzyme is cutting propperly, Make Kan+ plates, Work on making Cas9-VP16 biobrick
7/18/2013	KL	Transform EXPR_mKate-451s form LRs 7/17-KL
7/18/2013	MK	"Inoculation pDONR P4P1r
"
7/18/2013	CL	Ligated linearized U6 promoter we received from Samira with cr1,3,4, Digested px330 with BBSI and CIP, Gel extracted liberalized product and performed ligation reaction, Sent the px330 w/ cr1 and cr4 for sequencing, Sent px330 w/ cr1 samples 3,5,7,8,9 that cut with BBSI because the gRNA actually inserts a BBSI site, Miniprepped remaining six px330 w/ cr4 cultures
7/18/2013	BN	Digestion of Cas9 N-terminal backbone and then Golden gate with the linearized piece to create fusion vfp-cas9 proteins, Gel extract the linearized piece of Cas9,
7/18/2013	NH	Re-Goldengate VP16-Cas9, Analyze gels, Plan TRE_GAL4-VP16 dox ladder experiment, Plan out Cre recombinase experiments and update the experiment workflow. Also plan out the VP64 cas9 experiments
7/19/2013	KL	Pick colonies of EXPR_mKate-451s from 7/18-KL
7/19/2013	CL	Transformed about 400ng of DNA into 10g competent cells and plated 80uL of outgrowth on Kan plates
7/20/2013	HX	PCR, Gel extraction & Golden Gate: Acyl-TyA primers: GTW 025 & GTW 026 and Ngn2 primers: GTW 027 & GTW 028
7/21/2013	NH	Make miniprep cultures
7/22/2013	NH	Miniprep the Goldengate products, Figure out how the promoter situation, Restriction digest analysis, Send for sequencing, LR reaction if the digest analysis is okay
7/22/2013	HX	Transformed ENTR_L1_Acyl-TyA-mKate-His_L2; plated with beads on Kan plate; incubated overnight
7/22/2013	BN	Gel for extraction: Hyperladder I, U6 uncut, 5ul U6 w/ BBSI, px330 uncut, 5ul px330 w/ BBSI, U6 sample, px330 sample
7/22/2013	CL	LR C-terminal Cas9-splitvenus fusions to CAG promoter: L4_CAG_R1, L1_Cas9-VC155_L2, L1_Cas9-VN155_L2, L1_Cas9-VN173_L2, L1_eYFP_L2
7/23/2013	HX	Pulled plates out and inoculated 4 colonies in 4ml LB and 4ul Kan
7/23/2013	CL	Golden gate attempt 3 of N-terminal Splitvenus-Cas9 fusions: VN155-Cas9, VC155-Cas9, VN173-Cas9
7/23/2013	MK	Re goldengate things, BP reaction!
7/23/2013	NH/MK	Transform Goldengates, Transform U6_gRNA (Cr9), Finish and transform the BP reaction, Transfect w/ Hyo, Look at where I might be able to buy EXPR_Cre
7/24/2013	HX	Miniprep, Digestion
7/25/2013	NH	Plate the rest of the outgrowth from the goldengates on Kan+ plates, Rest of the time is for Design.
7/25/2013	LS	Re-try Golden Gate
7/25/2013	HX	Re-try Golden Gate
7/25/2013	MK	Pick colonies form the U6_gRNA, N-terminal Cas9 fusion golden gates, and BP reaction, Pick 10 colonies from each BP plate (BP 30, BP 5), Pick 5 colonies from one of the U6_gRNA(cr9) plates, Pick ALL of the colonies from each of the goldengates (GG 61, GG 63), BP's have Kan resistance, Goldengates have Kan resistance, U6_gRNA has Kan and Amp resistance
7/26/2013	NH	LR the goldengate products with the Hef1a and TRE entry vectors.
7/26/2013	MK	Miniprep the cultures for U6_gRNA, N-terminal Cas9 fusion goldengates, and BP reaction, Run analytical digest of all the miniprepped DNA!, Send for sequencing.
7/27/2013	CL	Digest and run gel of N-terminal fusion cut with BamHI and SalI: Cas9 for n-terminal fusion, VC155-Cas9, VN155-Cas9, VN173-Cas9
7/27/2013	NH	Midiprep the U6_gRNA(Cr9), N-terminal Cas9 fusions
7/28/2013	NH	Ligate in the 2xCr9 into the 2xCr9-CMV promoter construct (BP product)
7/29/2013	BN	Digest and run gel of CAG_cas9-splitvenus EXPR cut with BamHI and SalI: CAG_Cas9-VC155, CAG_Cas9-VN155, CAG_Cas9-VN173, CAG_eYFP,CAG_TagBFP, LR DEST 3-4
7/29/2013	MK	Transform DNA from LR, Mini prep BP, Midiprep U6, Digest new BP mini prep and old BP mini prep, Run restriction digest on the digests, Pick colonies from LR transformation
7/29/2013	HX	Restriction Map ENTR_L1_Acyl-TyA-mKate-His_L2
7/30/2013	NH	Redo LR
7/30/2013	HX	Redo Restriction Map
7/31/2013	NH	Transform LR
7/31/2013	HX	Transformed and plated colonies in preparation for a midiprep
8/1/2013	MK	Prepare miniprep culture of LR
8/1/2013	HX	Redo LR
8/2/2013	HX	Transformed second try of LRs
8/5/2013	MK	Set up midiprep cultures for the Hef1a_Cas9-Linker-VP16, Send the LR products to sequencing, PCR the BsaI on the 2xCr9-CMV promoter, Gel verify the PCR product
8/5/2013	NH	Goldengate the PCR product with the GG donor vector
8/6/2013	NH	Goldengate the PCR product with the GG donor vector, Send the LR products to sequencing, Column purify the pcr product
8/6/2013	MK	Set up midiprep cultures for the Hef1a/tre_Cas9-Linker-VP16 / also do cre plasmids
8/7/2013	NH	Midiprep the 5 cultures, Goldengate the PCR product and GG donor
8/7/2013	MK	Send LRs to sequencing
8/7/2013	MP	Blunt Cloning pGG_Acyl-TyA-Tev_DONR
8/8/2013	MK	Transform the Goldengate product / Plate, LR the things! (2xCr9-CMV with eYFP)
8/8/2013	LS	picked colonies and inoculate
8/9/2013	NH	Pick goldengate colonies, Transform and plate the LRs
8/9/2013	MP	Miniprep and mapping
8/10/2013	MK	Miniprep
8/12/2013	LS	Send Blunt cloning Tev3, 13, 19 for sequencing, digest 10ug of each one of them
8/12/2013	MP	Gel extract, ligation, plate on Xgal plate
8/13/2013	MK	Midiprep the 2xCr9-CMV_eYFP plasmid, Digest Goldengate products, Figure out what's going on with the 4xCr9-CMV_eYFP, Plan for the 2xCr9 -> 4xCr9:, Use gBlock 004 and digest with XbaI and EcoRI, Digest gBlock 33-2 with XbaI and EcoRI, Ligate the two and then send to sequencing, LR with EYFP.
8/13/2013	NH	Plan out Cas9 repression experiment, Cas9 activation experiment, Cre ladder v0.3, Design Cre/rtTA/L7aE primers.
8/14/2013	MK	Midiprep 2xCr9-CMV_eYFP, Run gels of the 2xCr9 fragments and other bits. (Gel failed)
8/15/2013	MP	cut with BsmBI, Run on a 1.5% gel
8/15/2013	MK	Midiprep the Cas9-VP16 constructs, Redigest the fragments with EcoRI-HF and XbaI in Cutsmart buffer.
8/19/2013	NH	Digest gBlock 002 and gBlock 33-2 with XbaI and EcoRI again

Summer Tissue Culture Lab Notebook

June 18th, 2013 Single Color Transfection of verified constitutive eBFP, eYFP, and mKate using Lipofectamine. Our goal was to increase our transfection efficiency. Transfected HEK293. (MP, KL, NH)

June 23rd, 2013 Single Color Transfection of verified constitutive eBFP, eYFP, and mKate using Lipofectamine. Our goal was to increase our transfection efficiency. Transfected HEK293. (NH, KL, JP)

June 26th, 2013 Single Color Transfection of verified constitutive eBFP, eYFP, and mKate using Lipofectamine. Our goal was to increase our transfection efficiency. Transfected HEK293. (NH, KL)

June 27th, 2013 Double Color Transfection of HEK293 verified constitutive eBFP, eYFP, and mKate using Lipofectamine. Our goal was to increase our transfection efficiency. (JP,NH, KL)

June 28th, 2013 Transfection of HEK293. Lipofectamine Optimization – We vary cell density, DNA concentration, and the amount of lipo to determine the optimal conditions for transfection. Unfortunately this transfection failed to yield any results. (NH)

June 29th, 2013 Transfection of HEK293. Lipofectamine Optimization – Lipo vs. the number of cells seeded. (KL)

July 1st, 2013 Transfection of HEK293. Prepped samples from 6.28 transfection for FACs and ran FACs. (CL) DNA/Lipo ratio transfection optimization using mKate and single color transfection of eBFP, eYFP, and mKate for basic microscopy. (JP)

July 2nd, 2013 Transfection of HEK293. Dox induction of a single fluorescent protein (Tre_eYFP).

July 3rd, 2013 Transfection of HEK293. Dox Ladder Single Color. Our goal was to create a graph of fluorescence vs. Dox concentration (KL)

July 4th, 2013 Transfection of HEK293 to verify Hef1A_eYFP-miR503 and TRE-t_eYFP-miR503.

July 5th, 2013 Prepped samples from 7.3 transfection for FACs and ran FACs. (CL) Practice transfection of HEK293 single and double colors. (MP, CL)

July 6th, 2013 Prepped samples from 7.4 transfection for FACs and ran FACs. (JP) Second transfection of the dox ladder in HEK293 (KL)

July 7th, 2013 Prepped samples from 7.5 transfection for FACs and ran FACs

July 9th, 2013 Transfected HK293 with Hef1a_Gal4VP16 and an eYFP with UAS target sites to show use of VP16 as an activator (NH)

July 10th, 2013 Single color transfection of Jurkats with LTX (KL). Use siRNA 451 and 503 on eYFP-4x451ts or eYFP-4x503ts in HEK293 to test receiver cell silencing (KL).

July 11th, 2013 Prepped samples from 7.9 transfection for FACs and ran FACs (NH)

July 12th, 2013 Prepped samples from 7.10 transfection for FACs and ran FACs (NH) HEK 293 practice transfection, single and double color (MP). Transfection of Acyl-Tya-GFP construct in HEK293 cells.

July 13th, 2013 Third repeat of HEK2093 Dox ladder transfection. Repeated Gal4-VP16 transfection.

July 14th, 2013 Prepped samples from 7.12 transfection for FACs and ran FACs (MP) Transfection of HEK293 with mKate with intronic miRNA and eYFP with miRNA target sites.

July 15th, 2013 Prepped samples from 7.13 transfection for FACs and ran FACs (CL) HEK293 transfection of traffic light reporter and Cas9 nuclease px335. Transfection of constitutively expressed split venus constructs with and without leucine zippers. (CL)

July 16th, 2013 Prepped samples from 7.14 transfection for FACs and ran FACs (NH). Transfection of HEK293 with Tre_Gal4VP16 with Dox (NH).

July 17th, 2013 Prepped samples from 7.15 transfection for FACs and ran FACs (KL) Repeated transfection of HEK293 with Acyl-Tya-eGFP (KL).

July 19th, 2013 Prepped samples from 7.17 transfection for FACs and ran FACs (KL) Repeated transfection of HEK293 with Acyl-Tya-eGFP (KL). Transfection of Tre_Gasl4VP16 with varying Dox levels.

July 20th, 2013 Single color transfection of Jurkats with LTX (KL)

July 21th, 2013 Prepped samples from 7.19 transfection for FACs and ran FACs (KL) Transfection of Jurkats with Acyl-Tya-eGFP using LTX (KL)

July 22nd, 2013 Prepped samples from 7.20 transfection for FACs and ran FACs (MP) HEK293 transfection with Hef1a_eYFP-503ts and mKate with intronic mi503

July 23rd, 2013 Prepped samples from 7.21 transfection for FACs and ran FACs (NH) Repeated Transfection of HEK293 with mKate with intronic miRNA and eYFP with miRNA target sites. Jurkat LTX Optimization.

July 24th, 2013 Prepped samples from 7.22 transfection for FACs and ran FACs (CL) HEK293 transfection of constitutively expressed split venus at different ratios (CL). Rh-PE staining of Jurkats (HX)

July 26th, 2013 Prepped samples from 7.24 transfection for FACs and ran FACs (CL) July 29th, 2013 Transfection of HEK293 with Ayl-Tya-GFP. Transfection of Jurkats with Neon.

July 30th, 2013 Rh-PE staining of Jurkats with new dye (HX)

August 1st, 2013 Transfection of constitutively Cas-9 split venus fusions without guide RNAs. Transfection of Cas9 nuclease targeting traffic light reporter

August 3rd, 2013 Prepped samples from 8.1 transfection for FACs and ran FACs (CL)

August 4th, 2013 Transfection of Tre_Gasl4VP16 with varying Dox levels. (NH) Repeated Transfection of HEK293 with mKate with intronic miRNA and eYFP with miRNA target sites.

August 6th, 2013 Prepped samples from 8.4 transfection for FACs and ran FACs (NH)

August 7th, 2013 HEK293 transfection testing the Cre-recombinase reporter (Addgene: pRLG3A - pLCMV_ECFP(loxP)(FRT)EYFP)

August 8th, 2013 Repeated Transfection of HEK293 with mKate with intronic miRNA and eYFP with miRNA target sites.

August 8th, 2013 Prepped samples from 8.4 transfection for FACs and ran FACs (HX)

August 14th, 2013 Transfection of LacI repression without miRNA repression.

August 21st, 2013 Transfection of Jurkats with eyfp with miRNA target sites using Neon (NH)

August 22nd, 2013 Transfection of HEK293 with eYFP with miRNA target sites and co-cultured cells with Jurkat exosomes.

August 23st, 2013 Prepped samples from 8.21 transfection for FACs and ran FACs (MP)

August 24th, 2013 Prepped samples from 8.22 transfection for FACs and ran FACs (NH)

August 26th, 2013 Prepped samples from 8.24 transfection for FACs and ran FACs (NH) Repeated Transfection of Jurkats with eyfp with miRNA target sites using Neon (KL)

Summer Tissue Culture Lab Notebook

September 5th, 2013 Transfection of HEK293 with eYFP with miRNA target sites and co-cultured cells with Jurkat exosomes.

September 7th, 2013 Prepped samples from 9.5 transfection for FACs and ran FACs (KL)

September 16th, 2013 Transfection of HEK193. Cas9-Venus fusions with target plasmid and guide RNA (CL)

September 17th, 2013 Transfection of HEK193. Cas9-Venus fusions with target plasmid and guide RNA (CL)

September 18th, 2013 Prepped samples from 9.16 transfection for FACs and ran FACs (CL)

September 19th, 2013 Prepped samples from 9.17 transfection for FACs and ran FACs (CL)

September 20th, 2013 Transfection of HEK193. Cas9-Venus fusions with target plasmid and guide RNA (CL)

September 21st Transfection of HEK293 with eYFP with miRNA target sites and co-cultured cells with Jurkats. (KL)

September 22nd, 2013 Prepped samples from 9.20 transfection for FACs and ran FACs (CL)

September 23rd Prepped samples from 9.21 transfection for FACs and ran FACs (KL)