Team:MIT/Notebook

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RNA Strand Displacement for Sensing, Information Processing,
and Actuation in Mammalian Cells

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 experimentation. During the 2012 spring semester, several members of our team started to learn different laboratory techniques from our instructors, and began some of the experimentation. Over summer, the full 16 member team - including one undergraduate student from another university - worked full-time on the project. Currently, during the 2012 fall semester, many 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
3/5/2012	RL	Restriction mapping pBACe with EcoR1
3/7/2012	RL	Made 1% agarose Gels
3/7/2012	RL	Ran gel of pBACe digest. NB. Make sure gel is in correct orientation
3/12/2012	RL	Ran gel again
3/14/2012	RL	Made antibiotic plates
3/20/2012	RL	Cultured competent Cells
3/27/2012	RL	pBACe 3,6 Gibson, electroporated into cells.
3/29/2012	RL	created primers for new pBACe3.6 experiment
3/30/2012	RL	grew cultures for miniprep
3/30/2012	RL	annealed andrew's oligions in PCR machine
4/2/2012	RL	Made and aliquoted SOC
4/4/2012	RL	Ran an analytic gel of 224 and 226.Hyperladder I, empty well, 226 G, 226 PI-SceI, 224 G, 224 PI-SceI, started gel at 100V at 9:40, run for 25 mins, finish at 10:05
4/10/2012	RL	Inoculated culture 1 from -80C iGem box using 1000:1 Amp (10 ul for 10 ml), split into 2 x 5ml tubes. Put in 37 C shaker at 9:00 PM
4/10/2012	EA	Made 1% agarose Gels
4/11/2012	RL	miniprep- CMV:Amcyan-miFF4 LacI colony 1. Practiced running a gel.
4/23/2012	RL	Used genious to model pBACe3.6 + J85224/226 forward and backwards to check for useful cut sites. For 224: Decided on NdeI to easily determine whether the part was inserted correctly or reversed.
4/25/2012	RL	For 226 it looks like either NcoI or NdeI would work - Probably go with NdeI for consistency with 224. Did them by hand in Genious for pBACe3.6 + 224. They do not form any hairpins and have Tms of 50 and 58 C Because the only variation between pBace3.6 + 224, 226, both forwards and reversed is in the section we're trying to sequence, these two primers should be sufficient to sequence each strand.
5/1/2012	RL	FACS training
5/7/2012	RL	Finished designing primers to check the insertions for the pBACe 3.6 gene. Some slight adjustments were made to correct for self annealing in the backwards primer as well as the melting temperature of the first. Starting work continuing the sequencing of pJAZZ. Reading Felix's notes and going over his sequencing progress before beginning.
5/12/2012	KG	Made 1% agarose Gels
5/15/2012	RL	More pJazz sequencing.
3/27/2012	AS	Diluted pUC 19 DNA to 50 pg/uL. Transfromed. Pepared gels.
4/1/2012	AS	Calculated competency of bacteria for transformation. Gel extractions. Made gels. Introduction to cell culture.
3/21/2012	NK	Prepared Z-competent 10G E.Coli using the Zymo protocol
4/4/2012	NK	Prepared LB + Agar for culture plates. Made 1% gels. Set up 4 PCR reactions for - PCR of J85224, J85226 with J85419, J85418 (To Gibson into PI-SceI site) - PCR of J85224, J85226 and J85430, J85431 (to add PI-SceI cutsites) .
4/6/2012	NK	DNA minipreps on qiacube.
4/9/2012	NK	Tissue Culture refresher with Ken.
3/13/2012	KB	Inoculate/Grow cells
3/14/2012	KB	Miniprep using QiaCube robot.
3/15/2012	KB	Gateway reaction of pENTR_L4_pCMV_R1:AmCyan-mirFF4-L2; Transformation
3/16/2012	KB	morning: check for colonies
3/19/2012	KB	Design restriction map and restriction digest the Input CMV and AM CYAN ; Start Culture for Gateway Products Miniprep
3/20/2012	KB	Gateway Product Miniprep ; Nanodrop
3/21/2012	KB	Restriction Digest of Gateway Product ; run gel of Gateway input and product
4/2/2012	KB	Restriction Digest of CMV promoter using Lincoln Lab's protocol ; Run gel ; Begin Gateway Reaction with new inputs
4/4/2012	KB	Making plates (3:00-3:40), Transformation of Gateway Reaction 2 (3:40-5:40)
4/5/2012	KB	morning: check for colonies (11:36 AM), afternoon: inoculate cells (4:11 PM)
4/9/2012	KB	Restriction Digest of (gateway input) pENTR_L1_AmCyan_mirFF4-2A-LacI_L2, Restriction Digest of (gateway output) pZDonor 1-GTW-2r-pCMV-AmCyan-mirFF4-2A-LacI (4:50 PM to 5:40 PM) ; gel electrophoresis (5:49 PM to 6:09 PM)
4/10/2012	KB	Redo Restriction digest of gateway input and 6 outputs (let digest sit for 3 hours) (3:30-7:00 PM) , gel extraction for Jon (4:00-5:00 PM)
4/11/2012	KB	Run gel 2X for gateway expression vector / AmCyan-LacI Input, 5 part Gibson Assembly, transformation, making gels
4/16/2012	KB	(3:00 - 6:00 PM) - Finish Restriction Digest of Minipreps of Culture 1 from Gateway of CMV (3-3:30): AmCyan-miRFF4-LacI for Keren, Observe Ken passage cells (3:30 - 4), Meeting with Giulio and Jameel (5-6)
4/17/2012	KB	Run Gel of CMV:AmCyan-MirFF4-LacI Digest (3:05-3:30 PM), Make Gels (5:20-5:45)
4/18/2012	KB	Inoculate pBACe3.6 colonies
4/20/2012	KB	Split HEK cells 1:3
4/23/2012	KB	Split HEK Cells 1:3 (3-3:30 PM)
4/24/2012	KB	Transfection of ROX+RNA gate and pEXPR Hef1a:Cerulean (5:00 PM - 8:00 PM), Split HEK Cells 1:6 (6-6:15 PM)
4/25/2012	KB	Split experimental culture 1:6, Microscopy of 4 Transfections from April 24th
4/26/2012	KB	(3:30 - 7:30 PM) - FACS of 4 transfections from April 24th, 5 new transfections, some microscopy, Split Culture
4/27/2012	KB	(4:15-4:40 PM) - Split HEK cells 1:12
4/20/2012	KB	Split HEK cells 1:6
5/2/2012	KB	Split HEK cells 1:3
5/3/2012	KB	Transfection of ROX with Ala'a, Split HEK cells 1:3
5/4/2012	KB	Split HEK Cells 1:12
5/7/2012	KB	Split HEK Cells 1:3
5/8/2012	KB	Repeat of 5 Transfections from April 26th, Split HEK Cells 1:12
5/10/2012	KB	Prepared Transfected Cells for FACS; Ran FACS with Deepak
5/15/2012	KB	Ran 5 Transfections again but with cells from Nathan, metafectine and a new annealed gate:output
6/11/2012	KB	Split cells 1:3 and 1:6
4/10/2012	KEK	Prepared for experiments by looking up details and becoming aquainted with machines available (plate reader). Resuspended Oligos
4/12/2012	KEK	Made serial dilutions of ROX part (1 uM - 1pM). Used Lu Lab Plate reader to measure fluorescence.
4/17/2012	KEK	Made 50 uL 100 uM aliquots of the RNA parts (S1, S6, Gate, Output). Annealed gate.
4/18/2012	KEK	Determined detection limit of plate reader via serial dilutions of DOX. Prepared 1x TAE 12.5 mM Mg2+ (filtered). We have quenching!
4/19/2012	KEK	Appear to have in vitro RNA strand-displacement.
4/24/2012	KEK	Plate reader in use
5/1/2012	KEK	FACS orientation, made 100 nM gate, secondary analysis of 4/26/2012 results. Performed a new kinetic experiment
5/3/2012	KEK	Took endpoint measurement for experiments from 5/01/2012. Tried strand displacement at 37 °C, but temperature dropped and stopped experiment. Made more 100 nM stock of gate:output. Set up ladder experiment protocol for plate reader.
5/8/2012	KEK	Annealed gate:output at RT (final concentration 45.45 uM) Annealed gate:input_sg at RT (final concentratin 45.45 uM). Annealed gate:input_s6 using the heatblock. Having issues observing strand displacement.
5/10/2012	KEK	Quantified gate:input anneals by measuring fluoresence vs. ROX. Didn't see any decrease in fluorescence -> the annealing protocol shouldn't affect fluorescence of ROX. Quantified RT-annealed gate:output by doing strand displacement.
5/13/2012	KEK	Set up annealing reactions on a plate reader for ROX concentrations of 2 nM, 5 nM and 10 nM. Measurements taken every 5 seconds for 30 minutes, then every 1 minute for <12 hours. Some datapoints for the latter will be missing -- another UROP needed to do OD measuremens every 30 minutes so I let him do that.
5/15/2012	KEK	Did a serial dilution of the wells from the strand displacement experiment to see if there a concentration effect to quenching (to answer the question whether we see increase in fluorescence when we dilute). Used ~10 nM sample from the plate, well G5. Made 5 nM, 1 nM, 100 pM, 10 pM dilutions with end-volume 100 µL.
5/17/2012	KEK	Plan: Do strand displacement reactions with the various 100+101 tubes that we have to make sure they work. Refiltered the 1X TAE buffer stock just in case - it's been a month since it was made.
6/8/2012	GA	LR reaction to make eYFP 4*luc expression vector
6/9/2012	GA	Transformed E.coli cells with EYFP 4*luc expression vector
6/10/2012	GA	Observed no colonies of transformed cells. Redo LR reaction to make eYFP 4*luc expression vector
6/11/2012	GA	Transformed E.coli cells with EYFP 4*luc expression vector made via the second LR reaction
6/12/2012	GA	Observed colonies. Stored in cold room
7/11/2012	RL, JE	LR Expression Vectors (Hef1A-eYFP-4xFF1) : Aligned sequence
	JK, WL	LR Expression Vectors (Hef1A-eYFP-4xFF1) : MIDIprep. Aligned sequence
	KG, JE	1. Hef1A/CMV-TetR: Transformed vectors into E.coli cells 2. FF4 construct: Transformed into E.coli cells
	JE,WL,	RNA Expression (U6-TetO-FF1): Performed Sequencing Alignment
	JK, RL	RNA Expression (U6-TetO-FF1): Inoculate for MIDIprep
	DI, LX	Pour Plates (IPTG/XGAL)
	AS	Miniprep GG donor vectors. Make cell stock
	DI, LX	Transform GG donor vectors
	RL, CV	Resuspend and anneal oligos
	LV, NS	PCR golden gate entry vectors
	NS, DI	PCR eBFP2 SP6
	KEK, WL	Plate reader study using Alexa- Em/Ex DNA Gate
7/12/2012	JK, KMG	Hef1A/CMV-TetR: Inoculate transformed cells
	JK	RNA Expression (U6-TetO-FF1): MIDI
	AS, EA	RNA Expression (U6-TetO-FF1): Restriction map/ send for sequencing
	WL	Pour more Plates (IPTG/XGAL)
	WL, DI, NS	Re-transform (DONR) Inoculate 50 mL (DONR)
	DI, NS	SP6: Gel Check and Gel purify
	KEK, WL	Plate reader study using Alexa- Em/Ex DNA Gate. Incubate with mRNA gates
7/13/2012	KMG	LR Expression Vectors (Hef1A-eYFP-4xFF1) : Take from cell stock and miniprep. Restriction map.
	RL	Send for sequencing Hef1A/CMV-TetR
	JK, AS	MIDIprep GG entry vectors
	DI	Transform GG entry vectors
	DI, NS	Use RNEasy kit to purify SP6
7/14/2012	JK	Inoculate for MIDIprep of Hef1A/CMV-TetR
7/14/2012	DI	Inoculate for Miniprep of GG entry vectors
7/16/2012	JK, DI	1.Design new LRs expression vectors 2. Hef1A/CMV-TetR ___Restriction Map (miniprep from 7/13), Send for Sequencing, Inoculate MIDI 3. Actuation: FF4 Decoys/TuDs: Design Restriction Maps (for all GG). Restriction Map (minipreps from 7/15). Send for Sequencing 4. eBFP2 1x-4x: Miniprep (inoculations from 7/15). Make Cell Stocks (of correctly restriction mapped samples)
7/16/2012	KEK, WL	Plate reader study of Alexa- Em/Ex DNA Gate
7/17/2012	DI, KEK	1. Design U6-S1, U6-S6 Expression vectors 2. Hef1A/CMV TetR: Restriction Map (miniprep from 7/13), Send for Sequencing 3. FF4: Restriction Map
		1. Re-design Restriction Maps (for all GG) 2. FF4 Decoys/TuDs: Restriction Map (minipreps from 7/16), Send for Sequencing 3. Make Cell Stocks (of correctly restriction mapped samples)
	DI, JK	Re-invitro transcribe SP6 contruct
	KEK, WL	Plate reader study: Alexa- Em/Ex DNA Gate. Incubate with mRNA gates
7/18/2012	KMG, JE	Restriction maps ( CMV tetR, FF4 constructs).
	CV, JK	In-vitro transcription of mRNA/ Redilute tetR entry vector. Set up new LR (CMV/Hef1A- TetR)
	DI, JK	Re IVT/Purify SP6-eBFP2
7/19/2012	KMG, JE, RL	U6-TetO - S1/S6: Get in Primers U6-TetO-S1/S6. PCR + RCR purify U6. GG + Transform U6
7/19/2012	CV, JK	CMV/Hef1A tetR: transformed into E.coli
7/20/2012	KMG, JE, RL	U6-TetO - S1/S6: Inoculate
7/20/2012	CV, JK	CMV/Hef1A tetR: inoculate transformed cells
7/21/2012	KMG, JE, RL	U6-TetO - S1/S6: Miniprep. Made cell stock
	CV, JK	CMV/Hef1A tetR: miniprep. made cell stock
	JK, LX	Inoculate (new GG) eBFP2 1x, eBFP2 2x, eBFP2 3x ,eBFP2 4x constructs
	DI, JK	Transform mKate-intronic FF4 (entry vector)
7/22/2012	JK, LX	Performed gel check of U6-TetO-S1 and U6-TetO-S6 to see whether we have the right constructs made
	KEK	Preliminary fuel studies
	DI,JK	1. Hef1A-TetR__ Miniprep 2. Decoy FF4-3p-4ntin __Miniprep, restriction map and sent for sequencing. Made cell stock. Inoculate transformed cells. Midiprep 3. TuD FF4-3p and TuD FF4-3p-4ntin __Aligned sequence and midiprep correctly aligned constructs 4. pEXPR_12_Hef1A:mKate-IntronicFF4__ transformed new LR 5. Transform mKate-intronic FF4 (entry vector)
7/23/2012	JK, LX	Miniprep eBFP2-1x, eBFP2-2x, eBFP2-3x, eBFP2-4x. Restriction mapped the constructs and sent them for sequencing.
	KEK	In-vitro study of fuel strand in the dynamics of strand displacement
	GA, FS	Checked nucleotide sequences for NOT gate
	DI, JK	1. Hef1A-TetR __ miniprep and map 2. Decoy FF4-3p-4ntin __miniprep. Map and sent for sequencing 3. TuD FF4-3p and TuD FF4-3p-4ntin __ miniprep 4. Q1_U6-TetO-FF4_QX __ obtained primers 5. mKate-IntronicFF4 (Entry Vector)__ miniprep from cell stock

7/24/2012	JK,LX	Align the sequencing results with our designs for eBFP2-1x, eBFP2-2x, eBFP2-3x, eBFP2-4x. Midiprep correctly aligned constructs
	KEK	Fuel studies. Measure and discard old plates
	GA, FS	Checked that domain X do not cause unwanted displacement. Ordered oligos and NOT gates.
	JE, KMG	Obtained HHR G-blocks
	DI, JK	1. Hef1A-TetR__ LR troubleshooting 2. Decoy FF4-3p-4ntin __ Aligned sequencing results. Midiprep 3. TuD FF4-3p and TuD FF4-3p-4ntin __ Aligned the sequencing results. Midiprep sequences correctly aligned
7/25/2012	DI, JK	1. Gradient PCR to check 2. Re-PCR Q1-eBFP2-QX constructs and purified the PCR products. 2. Re-annealed oligos. Performed gel check. Carried out GG cloning to assemble the parts for the constructs. Transformed the constructs into E.coli the identity of the constructs for U6-TetO-S1 and U6-TetO-S6. Gel check. 3. In vitro transcribed SP6-eBFP2 mRNA
	KEK	In-vitro study: mRNA as input (mN domains)
	DI, JK	1. Resuspended G-blocks. GG assembly and transformed the constructs 2. Re-anneal ALL oligos. Run gel to check 3. Discard all TetR Entry Vectors EXCEPT #4
	DI, JK	1. Hef1A-TetR__ carried out new LR reactions. Transformed. 2. Decoy FF4-3p__Inoculate and midi 3. TuD FF4-3p and TuD FF4-3p-4ntin __ Re-PCR Q1-U6-TetO-Q3, PCR Purify. Re-anneal oligos. Gel Check and GG annealed. Transformed. 4. Q1_U6-TetO-FF4_QX __ Gradient PCR. Gel check 5. pEXPR_12_Hef1A:mKate-IntronicFF4 __ new LR reactions. Transformed the products of the LR reactions. 6. pEXPR_12_Hef1A:eYFP-4xFF4__ new LR reactions. Transformed the products of the LR reactions. 7. Make new 5 uM primer stocks
	WL, AS	Re-dilute ALL canonical LR Entry Vectors to 5fmol stock solutions
	RL, LX	1. Send GG donor vectors for sequencing with M13 F & R. Send eYFP-4xFF4 from Box 2 for sequencing 2. Pour plates
	RL, LX, JK	Design an organized Workflow 0.2 for all of our correctly sequenced parts (Part name, concentration, DNA location, cell stock)
	EA, GA	Check Qiagen Inventory: Low on Wash Buffer PE for Minipreps. Low on Buffer N3 for PCR Purification. Enough tubes and buffers for this week, possibly next week. The kits should most likely be ordered beginning of next week
7/26/2012	JK,LX	1. PCR troubleshoot for U6-TetO-S1 and U6-TetO-S6 constructs. 2. Inoculate the transformed E.coli to amplify the constructs for eBFP2-1x, eBFP2-2x, eBFP2-3x, eBFP2-4x
	KEK	In-vitro study: NOT sensor design stock check. mRNA as input V2
	JE, KMG	Inoculate cells transformed with the GG constructs
	DI, JK	1. Hef1A-TetR__ Inoculate the transformed cells. New LR 2. TuD-FF4-3p and TuD-FF4-3p-4ntin- if GG/ anneals fail, order new primers 3. Decoy FF4-3p__ midiprep 4. TuD FF4-3p and TuD FF4-3p-4ntin __inoculate the transformed cells 5. Q1_U6-TetO-FF4_QX __PCR troubleshoot and ordered new primers 6. pEXPR_12_Hef1A:mKate-IntronicFF4 __ inoculate the transformed cells. New LR 7. pEXPR_12_Hef1A:eYFP-4xFF4__inoculate the transformed cells
7/27/2012	JK, LX	1. Ordered new primers for U6-TetO-S1 and U6-TetO-S6 constructs. 2. PNK treating of oligos (to add 5’P). Annealed oligos and GG transformed
	KEK	NOT sensor sequences. mRNA as input (appended sequence). PAGE purification of gate: outputs
	GA,FS	Troubleshoot NOT gate contructions
	JE, KMG	Miniprep the plasmids of cells transformed with GG constructs (HHR G-blocks. Mapped and send for sequencing. PCR (add SP6). Checked PCR on Gel. Inoculate and Midiprep
	DI, JK	1. Hef1A-TetR__ Transformed new LR 2. TuD FF4-3p and TuD FF4-3p-4ntin __PNK (to add 5'P) and GG annealed. Transformed. 3. pEXPR_12_Hef1A:mKate-IntronicFF4__ transformed new LR
7/28/2012	JE, KMG	Aligned the sequence of correct constructs. Midiprep. RNA gel
	JK, LX	Inoculate cells transformed with eBFP2 1x, eBFP2 2x, eBFP2 3x, eBFP2 4x constructs
	JE	Aligned sequence for HHR G-blocks
	AS	Inoculate cells transformed with Hef1A-TetR constructs +Variants (Ubc promoter)
	DI, WL	Inoculate cells transformed with pENTR_U6-TetO:TuD-FF4-3p and pENTR_U6-TetO:TuD-FF4-3p-4ntin (using old primers)
	RL, EA	1. Hef1A:mKateIntFF4 +Variants (Ubc, CAG promoter): Inoculate transformed cells 2. Hef1A:mKateIntFF5FF4: inoculate transformed cells 3. pEXPR_23_Hef1A-LacO:eYFP-4xFF4 AND pEXPR_23_TRE-tight-LacO:eYFP-4xFF4: inoculate transformed cells
7/29/2012	JK, LX	eBFP2 1x, eBFP2 2x, eBFP2 3x, eBFP2 4x constructs: miniprep, restriction map and make cell stock
	LX	SP6-eBFP2 DNA construct: check PCR on gel
	JE	HHR G-blocks: check PCR on gel
	AS	Mini prep and restriction map Hef1A-TetR constructs +Variants (Ubc promoter). Make cell stock
	DI, WL	Mini prep and restriction map pENTR_U6-TetO:TuD-FF4-3p and pENTR_U6-TetO:TuD-FF4-3p-4ntin (using old primers). Make cell stock
	RL, EA	1. Hef1A:mKateIntFF4 +Variants (Ubc, CAG promoter):Mini prep and restriction map. Make cell stock 2. Hef1A:mKateIntFF5FF4: Mini prep and restriction map. Make cell stock 3. pEXPR_23_Hef1A-LacO:eYFP-4xFF4 AND pEXPR_23_TRE-tight-LacO:eYFP-4xFF4:Mini prep and restriction map. Make cell stock
7/30/2012	KEK, CV	Send for sequencing NOT sensor
	JK, LX	eBFP2 1x, eBFP2 2x, eBFP2 3x, eBFP2 4x constructs: Send for sequencing
	LX	Gel purify SP6-eBFP2 DNA/ verify eBFP2 mRNA folding at room temperature
	GA, FS	NOT Gate: do gate annealing
	JE, KMG	Obtained NOT gate primers
	JE	Redo Gradient PCR of HH3 G-blocks. Check PCR on gel and gel purify
	AS	Hef1A-TetR constructs +Variants (Ubc promoter): Send for sequencing and inoculate for more colonies
	DI, WL	pENTR_U6-TetO:TuD-FF4-3p and pENTR_U6-TetO:TuD-FF4-3p-4ntin (using old primers): send for sequencing
	RL, EA	1. Hef1A:mKateIntFF4 +Variants (Ubc, CAG promoter): send for sequencing and inoculate for more colonies 2. Hef1A:mKateIntFF5FF4: Send for sequencing
7/31/2012	KEK, CV	experimental design of unmodified eBFP2 sensor
	JK, LX	U6-TetO-S1 and U6-TetO-S6 constructs: received new primers. Resuspend primers and PCR
	JK, LX	eBFP2 1x, eBFP2 2x, eBFP2 3x, eBFP2 4x constructs: Aligned sequencing
	LX	Purification of invitro transcribed SP6-eBFP2 mRNA
	KEK	tested new RNA G:O anneal, study mRNA sensor transfer curve and fuel transfer curve
	GA, FS	in vitro experiment to check the transfer function of NOT gate
	JE, KMG	Re-do PCR (HH3 G-blocks). Check PCR on gel and PCR troubleshoot
	AS	Hef1A-TetR constructs +Variants (Ubc promoter): aligned sequencing results. Miniprep (more colonies). Restriction map to check the constructs are correct. Make cell stock. Send for sequencing and inoculate cells for MIDIprep
	DI, WL	1. pENTR_U6-TetO:TuD-FF4-3p and pENTR_U6-TetO:TuD-FF4-3p-4ntin (using old primers): aligned sequencing data 2. pENTR_U6-TetO:TuD-FF4-3p and pENTR_U6-TetO:TuD-FF4-3p-4ntin (using new primers): obtained new primers/ resuspend primers. Anneal oligos-GG (with T4 kinase). Transformed cells
	RL, EA	1. Q1_U6-TetO-FF4_QX: obtained new primers, resuspend primers and PCR 2. Hef1A:mKateIntFF4 +Variants (Ubc, CAG promoter): Aligned sequencing results. Miniprep for more colonies, restriction map. 3. Hef1A:mKateIntFF5FF4: Aligned sequencing results
8/1/2012	JK, LX	Ordered Gblocks for U6-TetO-S1 and U6-TetO-S6
	LX	Invitro transcribed SP6-eBFP2 mRNA
	KEK	update old plate info and clean plate reader computer data
	GA,FS	planned invitro experiment to check the transfer function of NOT gate
	JE, KMG	HH3 G-blocks:Re-do PCR (gradient w/ Betaine), Check PCR on gel
	AS	Hef1A-TetR constructs +Variants (Ubc promoter): aligned sequencing results. MIDIprep and pass along to the transfection team
	DI, WL	pENTR_U6-TetO:TuD-FF4-3p and pENTR_U6-TetO:TuD-FF4-3p-4ntin (using new primers): inoculate transformed cells
	RL, EA	1. Q1_U6-TetO-FF4_QX:Grad PCR w/ Betaine, Check on gel and ordered gBlock 2. Hef1A:mKateIntFF4 +Variants (Ubc, CAG promoter): Miniprep (more colonies). Do restriction map to check the constructs are correct. Make cell Stock and send for sequencing. Inoculate the cels for Midiprep later on
8/2/2012	LX	PCR amplify SP6-eBFP2 DNA and do RNA purification of SP6-eBFP2 mRNA
	JE, KMG	HH4 G-blocks:Re-do PCR (gradient w/ Betaine), Check PCR on gel
	DI, WL	pENTR_U6-TetO:TuD-FF4-3p and pENTR_U6-TetO:TuD-FF4-3p-4ntin (using new primers): Miniprep. Restriction map to check the constructs. Make cell stock and send for sequencing. Inoculate transformed cells for MIDIprep later on
	RL, EA	Hef1A:mKateIntFF4 +Variants (Ubc, CAG promoter): Miniprep (more colonies). Aligned sequencing data and MIDIprep. Pass along MIDIprep samples for the transfection team
	DI	Designed new T7 IVT Primers (eBFP2,LacI,LacZa, HH's) and ordered primers
8/3/2012	DI, WL	pENTR_U6-TetO:TuD-FF4-3p and pENTR_U6-TetO:TuD-FF4-3p-4ntin (using new primers): Align Sequencing results and Order gBlocks
8/6/2012	KEK, CV	1. NOT Sensor: ordered sequences and simulate 2. unmodified eBFP2 sensor: obtained new fuels, anneal domain 1 G:O and run experiment to simulate
	LX, JK	eBFP2 1x, eBFP2 2x, eBFP2 3x, eBFP2 4x constructs: Ordered new primers and Gblocks
	LX	SP6-eBFP2 DNA: check products on gel. Gel purify
	KB	Ordered 2' O-me RNA intermediate gates
	KEK	Obtain new primers, Resuspend primers and PCR the respective templates
8/7/2012	KEK, CV	unmodified eBFP2 sensor: troubleshoot, look at overnight data, revise plans. get more mRNA. compare domains (run experiment)
	LX	1. U6-TetO-S6: Resuspend new Gblocks. GG anneal. Transformed cells with constructs 2. SP6-eBFP2 mRNA: IVT RNA purification
	KEK	design gate:outputs to be transcribed in vivo
	GA, FS	NOT Gate: experiment to test transfer function at higher concentration
	JE, KMG	Gradient PCR HH2 G-blocks
	RL, AS	1. Hef1A:TetR-KRAB Expression Vector: transformed cells with vector 2. Q1_U6-TetO-FF4_QX: resuspend new g-blocks. GG anneal and transformed cells with constructs.
8/8/2012	KEK, CV	1. NOT Sensor: anneal 2. unmodified eBFP2 sensor: look at overnight results of debug run and overnight results of domain comparison. Rerun experiment/Make new plan
	LX	1. U6-TetO-S6: inoculate transformed cells 2. U6-TetO-S1: resuspend new Gblocks. GG anneal and transformed cells with constructs 3. SP6-eBFP2 mRNA: IVT RNA purification 4. PCR T7-eBFP2 DNA
	KEK	in vitro test of 2'O-Me parts
	RL, AS	1. Hef1A:TetR-KRAB Expression Vector: inoculate cells (TE buffer). Miniprep. Make cell stocks. Restriction map and pass long constructs to transfection team 2. Q1_U6-TetO-FF4_QX: inoculate transformed cells
8/9/2012	KEK, CV	1. NOT Sensor: run experiment 2. unmodified eBFP2 sensor: look at overnight results and analyze
	LX	1. U6-TetO-S6: miniprep. restriction map. send for sequencing. make cell stock and inoculate more cells for MIDIprep 2. U6-TetO-S1: inoculate transformed cells 3. T7-eBFP2 DNA: check product on gel and gel purify
	KEK	discuss about bulges and transcribing gate:output as a single strand
	RL, AS	1. pENTR_U6-TetO:TuD-FF4-3p and pENTR_U6-TetO:TuD-FF4-3p-4ntin (using new primers): obtained and resuspend new G blocks. GG anneal and transformed cells 2. Q1_U6-TetO-FF4_QX: miniprep. restriction map. send constructs for sequencing. Make cell stock and Inoculate more transformed cells for MIDI
	JE	Ordered new reverse primers and Inoculated new minipreps
8/10/2012	LX	1. U6-TetO-S6: aligned sequencing results. MIDIprep 2. U6-TetO-S1: Miniprep. Restriction map. Send for sequencing. Inoculate transformed cells for MIDIprep 3. T7-eBFP2 mRNA: invitro transcribed and RNA purification
	JE	miniprep HH2 G-blocks
	RL, AS	1. pENTR_U6-TetO:TuD-FF4-3p and pENTR_U6-TetO:TuD-FF4-3p-4ntin (using new primers): inoculate transformed cells 2. Q1_U6-TetO-FF4_QX: Aligned sequencing results. MIDIprep
	KEK	Testing new anneal protocols
8/11/2012	RL, AS	pENTR_U6-TetO:TuD-FF4-3p and pENTR_U6-TetO:TuD-FF4-3p-4ntin (using new primers): miniprep, restriction map. Send for sequencing and inoculate cells for MIDI prep
8/11/2012	LX	U6-TetO-S1: Aligned sequencing results. MIDIprep
8/12/2012	RL, AS	pENTR_U6-TetO:TuD-FF4-3p and pENTR_U6-TetO:TuD-FF4-3p-4ntin (using new primers): aligned sequencing results. Make cell stocks. Inoculate cells for MIDIprep
8/13/2012	JK, LX	Ordered new primers/ gBlocks for eBFP2 1x , eBFP2 2x, eBFP2 3x and eBFP2 4x
	KEK	New RNA anneals
	GA, FS	Plan in-votro experiments for strand displacement using NOT gate
	JE	HHR G-blocks: check PCR on gel
	RL, AS	pENTR_U6-TetO:TuD-FF4-3p and pENTR_U6-TetO:TuD-FF4-3p-4ntin (using new primers): send for sequencing.
	KEK	Testing new rRep6, dsROX
8/14/2012	KEK, CV	debug annealing problems and carried out transfer function experiment for NOT sensor
	LX	In-vitro transcription of T7-eBFP2 mRNA
	JE	HHR gBlocks: Check other 3 hammerheads on gel. Purification showed low concentration. Redo PCR amplification with T7 promoter
	RL, AS	pENTR_U6-TetO:TuD-FF4-3p and pENTR_U6-TetO:TuD-FF4-3p-4ntin (using new primers): Aligned sequencing results, MIDIprep. Re-inoculate cells for MIDIprep
	KEK	Testing dRep6 consistency with respect to their positions on the plates, observed dRep6 coming apart, carried out positional analysis
8/15/2012	KEK, CV	1. Do literature search for mRNA target: how does temperature affect dRep6/ Tested 2'OMe RNA for mRNA sensing (at 37C) 2. Test with mRNA for NOT sensor 3. Do transfer function experiment for unmodified eBFP2 sensor
	JK, LX	Make template for T7/SP6 lacI/lacZ DNA, mRNA
	JE, KMG	1. Retry purification of HHR gBlocks 2. Design new primers for HHR with Stem and mKate
	RL, AS	pENTR_U6-TetO:TuD-FF4-3p and pENTR_U6-TetO:TuD-FF4-3p-4ntin (using new primers): Midiprep and pass long to transfection team
	KEK	Testing fuels, addition of inputs in a step-wise manner
8/16/2012	KEK, CV	Design new reporter/ test with mRNA for NOT sensor and carried out transfer function experiment for unmodified eBFP2 sensor
8/16/2012	JE, KMG	Ordered new primers/ gBlocks for HHR with stem and mKate
8/17/2012	KEK	Testing Rox on 384 plate/ Running reactions in test tubes and taking samples from tubes at different time intervals
8/18/2012	KEK	Anneals/ dsROX vs rRep6, testing the effect of carrier RNA
8/19/2012	KEK	Testing positional variants on 384, newest PCR anneal
8/20/2012	KEK	Dose response for S1 RNA carrier, dRep6 PCR anneal testing. Testing PCR Gate-Outputs, mRNA sensing
9/7/2012	KEK	annealed reporter
9/20/2012	KEK	new long anneals
9/27/2012	KEK	annealed and tested the newest RNA strand design

Spring Tissue Culture Lab Notebook

April 9th, 2012

We are using HEK cells with constitutive EYFP. Typically, they need to be passaged 2-3 days, today we passaged at 1:6 ratio.

April 11th, 2012

Split at a 1:6 ratio. They will need splitting again by Friday. Also, iGEM now has P1000 tips and culture dishes in the bottom drawer. All of our goods are labeled "iGEM." The P1000 tips are to be used with the aspirator when aspirating liquids in the hood. Be sure to EtOH before and after use. Last night (4/11/12) Kenneth metafected cells at 11 pm. Movies are here: http://www.youtube.com/playlist?list=PL07C09A5037691E54

April 13th, 2012

Split at 1:12 ratio, so they will be able to last 3 days (till Monday). Made 10 Aliquots of Typsin (10 mL). They are on the bottom shelf of the freezer, labeled with iGEM on them. When you need to passage cells, take an aliquot and thaw in the hot water bath. For now, we won't be needing all 10 mL in the aliquot so I need to check if we should freeze it or put in the fridge.

April 16th, 2012.

Made media according to the formula above. Katie split cells today at 1:6. They need splitting on Wednesday. Also, materials for making media (except DMEM, and essential AA's), will be stored in aliquots in the freezer, labeled. Essential AA's will be aliquotted and stored in the +4. To make media, you will simply need remove the 50 mL of DMEM, and place that into a labeled conical tube. Then add thawed frozen components and essential AAs. Frozen components are on the bottom door shelf of the freezer. Essential Amino Acids are in the fridge on the door as well. DMEM can be found in the cold room, some is stocked in the metal min-fridge in the back too.

April 18th, 2012

Split cells at 1:6 ratio. Needs to be split again Friday. Also expanded what we have in culture. A back up dish will be maintained by myself only. Another back up dish will be maintained by Katie only. The plate labeled for experiments can have cells taken from it for transfections etc. When this is used, please notify me, so I can replace it.

April 20th, 2012.

Katie and I (Nathan) split cells today at a 1:13 ratio. They need to be split again on Monday. April 23rd, 2012. Nathan split his cells 1:6 (the petri dish labeled NK and the one labeled experiment) and will split again on Wednesday Katie split her cells 1:3 to prepare for transfection tomorrow Followed same protocol as above.

April 24th, 2012.

Katie used 2 mL of her cells that she did not use for transfection to split in a 1:6 ratio (2 mL cells, 11 mL of media) following standard protocol Katie also performed two transfections: (1) ROX+RNA(Gate) + CFP into her HEK293 cells (2) just CFP into her HEK cells with the help of Kenneth. Deepak is assisting with the confocal, the Zeiss and FACS.

April 25th, 2012.

Katie split the experimental plate 1:6 at 7:30 PM. Need to be split again on Friday. Nathan split a backup plate around 3 pm. Need to be split again on Friday.

April 26th, 2012.

Katie performed 5 transfections (using her culture). Katie also split her cells 1:3. They need splitting tomorrow as well as do Nathan K's and the experimental plates.

April 27th, 2012.

Nathan split a Back up and experimental plate were both split at 1:12 Katie split her plate 1:12.

April 30th, 2012.

Katie split her plate 1:6 in preparation for Ala's Transfections on Wednesday.

May 2nd, 2012.

Katie split her plate 1:3 in preparation for Ala's Transfections on Thursday.

May 3rd, 2012.

Katie performed 3 transfections of ROX with Ala'a. One with 0.1 ug ROX, one with 1 ug ROX and one with 3 ug ROX. Each also with 5 uL of CFP. Katie also split HEK cells (labeled KB) 1:3. Will split again on Friday

May 4th, 2012.

Nathan split cells 1:12. Will be ready by Monday. Will probably need to make another bottle of media by Monday. Katie split cells 1:12. Will be ready by Monday.

May 7th, 2012.

Nathan split cells 1:6. Will be ready by Wednesday. Also made a new bottle of media. Its labeled iGEM, Fully supplemented (with L-glu, Non-Ess. AA, FBS, P/S), and 5/7/12 Katie split cells 1:3. Will be ready tomorrow for transfection

May 8th, 2012.

Katie split cells 1:12. Katie also did 5 transfections (repeated from April 26th)

May 9th, 2012.

Nathan disposed of Katie's cells in culture. Split two plates 1:6.

May 11th, 2012.

Reducing what we have in culture to one plate. Split 1:12, will be ready on Monday.

May 14th, 2012.

Cells were quite confluent (95%), and split on the lower side of ~1:6 (2 mL from 13 mL total). Will be ready by Wednesday Also seeded a plate 1:3 for Katie, which will be ready by Tuesday to use in transfections. Also made more trypsin aliquots.

May 15th, 2012.

Katie used Nathan's cells that were seeded 1:3 for 5 transfections. All Details on [RNA & ROX Lifetime Experiment|igem2012:RNA & ROX Lifetime Experiment] page

May 16th, 2012.

Cells in dish marked NK were underconfluent. Possible that they were either split too hard, over trypsinized, or over confluency was no good. Media color is fine (Red), so will come back tomorrow and see if they need to be split or not. Also need to get them back onto a MWF splitting schedule. New aim is to re suspend in 12 mL total to make 1:6, 1:12 splits more straightforward. Plan is to split 1:3 Thursday, which means they would be confluent by Friday. Then split 1:12 for over the weekend. This of course depends on the confluency reached by tomorrow.?Could also do a split that is 1:24 (which would last 4 days roughly before splitting), and see how that turns out by Monday.

May 17th, 2012.

Cells in NK dish were properly confluent (80%). Seeded two plates, one at 1:3, which will then be split tomorrow at 1:12. The other 1:24, and to be safe/relatively consistent with the other plate, will do a media change tomorrow. By Monday, going to check which looks more viable in terms of media colour, confluency etc. Then going to decide which to keep. (Serial splits, or one large split)

May 18th, 2012.

Cells in 1:3 were confluent... Pipetted up and down to detach from the plate instead of trypsin (don't want to loose healthy cells that may not have adhered). Final volume was 10 mL, so split 1:10. I checked under the scope before placing in the incubator, and they look similar to the 1:12 splits, so they should last until monday. To be safe, will check on Sunday their confluency. 1:24, changed media. But it is possible that some cells were washed away in the process (especially if they didn't adhere). Will check sunday as well, but the populations looked incredibly low. It may make sense for the summer to bring up a new stock from the freezer (depending on how high passage numbers are getting from my plate, which is all we have in culture now).

May 19th, 2012

Both cell populations looked incredibly low. The 1:24 split obviously has less cells than the serial split. However, neither were even 50% confluent, and the media still looks rather healthy. Will probably check on Monday for media color and see if a media change is necessary.

May 21st, 2012

Both cell populations looked low, but the serial split plate looked better than the 1:24. Changed media in the 1:24. Trypsinized the serial split to break up the large clumps of cells to spread more evenly in the plate, but did not split at a ratio (13 mL of cell solution went back into the dish). Will check on Wednesday.

May 23rd, 2012

Serial split plate was destroyed, none of the cells adhered/recovered from trypsinization. 1:24 plate has low populations, but looks OK. Did a media change, will check again on either Thursday afternoon or Friday.

May 25th, 2012

Media color is normal, but not changing at all. Cells are alive, but they are not spreading out on the plate. More or less forming "colonies." Today, they were trypsinized to break up the colonies, spun down at 260g (according to this Penn iGEM protocol ) for 5 minutes and then resuspended in fresh media. The entire solution was placed into the dish. Will check tomorrow afternoon their confluency. At this point, if these cant come back it would be wise to get a fresh stock of 293Ts. We also should make frozen DMSO stocks so that when mistakes occur, we can at least work from the same passage.

May 26th, 2012

Cells in culture look good! Growing more as a monolayer and not colonies. Looking about 70%-75% confluent. Will split 1:6 today (out of 12 ml) and check again on Monday. Hopefully we can get them back on the MWF schedule. Also as an extra precaution, cells were centrifuged out of the trypsin/media mixture and resuspended in 12 mL fresh media prior to seeding two tissue culture dishes.

May 28th, 2012

Cells look good. Both dishes were split 1:6 (out of 12 mL). Followed the normal protocol (no centrifuging). During the protocol, there was a problem with the pipette aid. It would aspirtate, but not dispel fluid. Had to grab a pipette aid from the hood next to the microscope (washed with EtOH when transferring to and from hoods). Emailed Patrick about it.

May 30th, 2012

Patrick emailed over info about what was going on with the pipettor in the hood. Cells are growing, but a bit underconfluent (50%-60%). Since I can not come into lab tomorrow, going to split 1:6 anyways, check on Friday. If still underconfluent a bit on Friday, will let go to Saturday. I would rather have the cells be underconfluent and in log phase than overconfluent and in a lag phase.

June 1st, 2012

One plate was allowed to grow for an extra day. The other was split 1:12, into two dishes to last until Monday (which they will be back at ~60% confluency). This gives us a backup plate in the event that letting a plate go an extra day means the cells are over confluent.

June 2nd, 2012

The plate that was allowed to go the extra day looks good, 80%-90% confluent, and not overgrown at all. Will Split 1:6 today, which will put back on MWF splitting schedule. Cells split yesterday will be checked on Monday... Could do either a 1:3 split from the backup plates on Mon. to get the 80-90% confluency by Wed. or simply expand our cultures from the one plate that looks good (80-90% confluent as of today). Expanded cultures from the properly confluent plate.

June 4th, 2012

Split cells 1:6. Will check on Wednesday.

June 6th, 2012
Made a new bottle of media. Split cells 1:6. Will check on Friday.

June 8th, 2012
Cells were underconfluent and allowed to grow an extra day.

June 9th, 2012

Cells are still underconfluent. Seeded a dish 1:3, the other 1:6. To ensure an accurate 12 mL final volume, cells in the trypsin/media mixture were centrifuged at 260g for 5 mins, and then resuspended in fresh media prior to seeding. Also, I dropped our aspirating tool on accident, and the glass portion to it broke. Put it in the pickle jar sharps container. Will need to make another one, or simply use pastuer pipettes. Also received an important email from Kenneth regarding the incubators. Information from said email is below. The water tray in the bottom of the incubator needs to be checked and filled when empty. Without water, the incubator will not be at the proper humidity and the CO2 sensor will not work properly, which means no CO2 and the media becomes too basic (purple) and cells will die/grow slowly. You can use the DI water from the white tap on the sink, no need to add anything to the water. Also, use a graduated cylinder to make easier! (need to check about antibacterial blue stuff)

Summer Tissue Culture Lab Notebook

Week One: 6/11/12 - 6/17/12

June 11th, 2012

One plate of cells was underconfluent, will be ready by tomorrow (the 1:6 from 6/9/12). The 1:3 plate was 90% confluent. Seeded two plates at 1:3, two plates at 1:6, to have cells ready by tomorrow and Wednesday. The plan is to potentially use one plate tomorrow to seed more plates for Thursday and Friday, and expand what we have in culture. We need to have a conversation with Deepak to have a concrete plan on how much we need to expand our cultures.

June 12th, 2012

To Do: 1cm plate for Deepak/ other students both 1:6 plates appeared way under confluent on the scope (about 10% confluence) used 1:3 plate from 6/11/12 for transfections of pEXPR Hef1a:Cerulean and IGEM-BBa_K779100 (the gate i.e. the fluorophore + RNA)

June 13th, 2012

Cells are still underconfluent, and have reached a pretty high passage number (at least 30). So, we are talking about getting fresh cells from the summer, and this time making a DMSO stock so we can return to a low passage. We performed 6 transfections in duplicate using both 1:6 plates.

June 14th, 2012

Bleached and threw out all remaining 1:3 and 1:6 plates which we were maintaining Received a frozen DMSO stock of HEK293 (without constitutive YFP) from Kenneth (1.5x 10^6 cells). Going to make more DMSO stocks on Monday.: We thawed out the cell. Added them to 12 mL of DMEM with phenol red. Spun down at 1180 RPM for 4 minutes. Aspirated off the media / DMSO. Resuspended the cells in 12 mL of fresh DMEM. Transferred all 12 mL of culture onto a new dish. We also observed the Zeiss data from the transfections.

June 16th, 2012

As of today, we don’t have HEK+EYFP, but we may not need them Split HEK293 today 1:6 for monday. Seeded 5 plates, so that we have enough for transfections and to create DMSO stocks. Also added DIH20 to the tray at the bottom of the incubator, to keep humidity and CO2 levels constant. Also created more FBS stocks. One for Freezing Media, another for making media for normal culture.

Week Two: 6/18/12 - 6/24/12

June 18th, 2012

We split one plate of cells and seeded two dishes 1:6. One is back up, the other to potentially use for transfections on Wednesday. We also seeded a dish 1:3 to use in transfections tomorrow for imaging. We used three plates to make cell stocks of HEK 293T. 3 cryovials contain 1.2x10^6 cells. 3 cryovials contain 1.5x10^6 cells. One plate was then used for transfections for FACs. A static image was also taken.

June 19th, 2012

We seeded one plate 1:6 to be ready by Thursday. We also repeated the transfections for FACs analysis and Continuous imaging.

June 20th, 2012

Prepared samples for FACs analysis. We also split one plate 1:6 to have two dishes ready by friday for transfections. Today we also transfected eCFP to get a quantitative measurement of our transfection efficiency on Friday.

June 21th, 2012

Split 1:6 plate from 6/19 1:6 to be ready for Saturday.

June 22nd, 2012
split 1:6 two plates for Sunday (possibly Monday)

June 24th, 2012
There were HEK293+EYFP in our incubator, dated 6/22. As a result, the cells were very confluent, almost 100%. Cells were seeded at 1:3, 1:6 and 1:12. Hopefully, they are still viable. The 1:3 plate, if totally confluent again tomorrow can serve for cells tocks, the rest for transfections if necessary through the week. Or, making more cell stocks... Today I am also bringing up one of the cryovials of HEK293 to test cell viability. Another bottle of fresh media was made. The concentrations are slightly off, by around 1.25%, but according to Deepak it should be OK. Also autoclaved more pastuer pipettes.

Week Three: 6/25/12 - 7/1/12

June 25th, 2012

Made 5 cryovials of HEK293+EYFP.

June 27th, 2012

corrected media today. FBS was at 8.8%, so we created another bottle of media at 11.2%, then combined and filter sterilized together. Made FBS aliquots.

June 28th, 2012

Cells more confluent with new 10% media! Split six 1:6 plates for Saturday. Need a P1000 in the hood and need P20 calibrated

June 29th, 2012

Split two 1:6 plates of HEK293 for Sunday and two 1:6 plates of HEK293 eYFP for Sunday. Aliquoted more FBS. Will need more media soon ( we have another 350 mL bottle...) Got a new calibrated P20! Also aliquoted more trypsin.

June 30th, 2012

Split two 1:6 plates of HEK293 for Monday and two 1:6 plates of HEK293 eYFP for Monday. Also seeded a 6 well plate for FACs training. Should have 200,000 cells for each sample by Monday. Also did a media change on the ROX transfection, and took some images using the microscope in the TC room. Saved on the flashdrive there. Accidentally spilled bleach while cleaning the vaccum trap. Contained with paper towels, and cleaned up. (only spilled around 5-10mL). Also, our box of short pastuer pipettes is low, will need to autoclave more soon.

July 1st, 2012
Split two 1:6 plates of HEK293 for Tuesday and two 1:6 plates of HEK293 eYFP for Tuesday. Also seeded another 6 well plate for FACs training, but this time a higher density. Will probably pool samples for tomorrow. Should have ~1,000,000 cells for between the two dishes for tomorrow. Disposed of old (turning basic) media. Did FACS analysis and imaging of the ROX transfection that compares RNAiMax and Lipo2k.

Week Four: 7/2/12 - 7/8/12

July 2nd, 2012

Split two 1:6 plates of HEK293+eYFP for Wednesday. Split two 1:6 plates of HEK293 for Wednesday. Made a new 500 mL bottle of DMEM with phenol red with 10% FBS, 1% Nonessential Amino Acids, 1% L-Glutamine, 1% Pen/Strep

July 3rd, 2012

Split two 1:6 plates of HEK293 for Thursday. HEK293+EYFP were a bit under confluent. Also brought up a frozen stock of HEK293+EYFP to test cell viability.

July 4th, 2012

Split two 1:6 plates of HEK293 and HEK293+EYFP for friday.. HEK293+EYFP were a bit under confluent, let go till tomorrow. Did a media change on the transfections. Transfections in phenol red media were not as intensely red as before, only slightly tinted. Imaged and ran FACS analysis on transfections. Seeded a 6 well plate with ~ 500,000 cells per well for FACS training.

July 5th, 2012

Split two 1:6 plates of HEK293 and HEK293+EYFP for Saturday. Cells brought up from frozen stock on 7/3/12 about 70% confluent. Will also check again on 7/6/12. Ran FACS Analysis on Transfections from 7/3/12. Received Alexa tagged output for our Rep6. Diluted and annealed with the ROX tagged gate. Will transfect tomorrow. Also expecting LRs to transfect tomorrow.

Week Seven: 7/22/12 - 7/29/12

Sunday July 22nd:

Maintenance: We froze down 10 stocks of HEK293's at P3, which are in the -140. We have one plate of HEK293+eYFP split 1:6 today to be ready Tuesday. We have four plates of HEK293's split 1:6 for Tuesday. We have four plates of HEK293's split 1:3 for Monday. We have one plate of HEK293's split Friday ~1:6, which were under confluent, which should be ready tomorrow. Need to bring up one stock of 293's on Thursday to check for viability. Also need to remember to seed HEK293+eYFP's for Jonathan for the 26th. Also need to figure out the proper amount of L-Glutamine to add to the clear media. Prepared KB's Optimizing efficiency of dsRNA + DNA samples for FACS.

Monday July 23rd:

Split 4 plates of HEK293s 1:6 Ran FACS on KB's ALEXA, ROX, ALEXA:ROX, +S1, +S6 experiments (following preparation protocol from above)

Tuesday July 24th:

Took one plate of HEK293's at P4 (that were split 1:3) to freeze down into 10 cryovials, with ~1,000,000 cells each Transfections: KB repeated ALEXA/ROX transfections with 24 well plates (details on expt page) NK repeated the rTTA3 system, this time with a DOX curve Passaging: Split four plates of HEK293s at 1:6 for Thursday Split one plate of 293+eYFP for Thursday FACS Prep: Seeded 250,000 cells per well in a 6 well dish for Jon and whoever else wants FACS training on Thursday Seeded 2 wells of HEK293s, 2 wells of HEK293+eYFP, 2 wells of half 293s & half eYFPs. Will prep for FACS on Thursday

Wednesday July 25th:

1. Moved frozen down cells from -80 to -140 (at 9:00 A.M.) Note: Next Tuesday we should bring up a vial to test viability - KB - 7/26 2. Performed a media change on 9 wells of KB's Alexa, Tag transfections from 7/24 (at 10:30 A.M) 3. Supplemented Red Media (12:00 PM): 4. Supplemented Clear Media (12:00 PM): 5. Supplementing Media Notes: Keep all components in small aliquots - easy to thaw Always aliquot the entire FBS bottle - it is bad to keep thawing/freezing FBS 6. Eta transfected constitutive colors at 2:00 PM for Friday Microscopy Training 7. Katie transfected Alexa:ROX into her Tag well at 2:30 P.M. to optimize dsRNA + DNA transfection 8. Linh retransfected the eYFP knockdown system with FF1 to show that her efficiency and results are repeatable 9. Nathan induced a DOX ladder to his rtTA3/DOX system and also retransfected the entire system (due to volume issues the previous day) 10. Split 5 plates of HEK293's 1:6

Thursday July 26th:

7:40 - 8:40 A.M. - Prepare Katie's ALEXA FACS samples and Nathan's DOX FACS Samples 10:45 A.M. - 12:00 PM - FACS KB and NK samples 2:00 PM - NK: Prepare FACS samples for JE + AS We prepared 2 wells of 250k HEK293s. 2 wells of HEK293s+EYFP. 2 wells of mixed both 293 and 293+EYFP 4:30 PM - KB: Transfect S6 Input annealed to gate with Hef1a:eYFP Marker 4:45 PM - KB: Split four plates of HEK293's 1:6. Split one plate of HEK293+eYFP 1:6 5:00 PM - Nathan induced DOX ladder on his rtTA3 transfections

Friday July 27th:

8:00 AM - LV + NK prepare FACS samples 11 AM - FACS NK's rtTA3 DOX, LV's FF1 KD 2:00 PM - KB Transfect Alexa:ROX, +S1, +S6 system with 2X input. Transfect S6:Gate to well transfected with Hef1a:eYFP yesterday. • Protocol - Transfection72712.xlsx 3:30 PM - LV Transfect miRFF1 - eYFP knockdown system 4:30 PM - NK transfect three plasmid / four plasmid constitutive colors to up efficiency. Let's break 90%! Also retransfect DOX curve system? 6:00 PM - KB passaged four plates of HEK293's 1:6 (to be ready Sunday)

Saturday July 28th:

FACS KB Optimization / 2x Input Experiments FACS EA's Single Plasmid Constitutive Colors Expt. Induce DOX (NK) Transfect multi plasmid efficiency (NK) Passage

Sunday July 29th:
* NK DOX FACS * LV Knockdown FACS * EA transfect constitutive colors for scope / FACS training * Passage

Week Eight: 7/30/12 - 8/5/12

Monday July 30th:

* FACS NK multi plasmid efficiency
* LV transfect FF1 KD with different parameters
* KB transfect ALEXA/ROX system with 3X input. Also try dsRNA + ssRNA optimization as suggested by Eerik.
* Passage

Tuesday July 31st:

* Confocal KB's transfection
* NK transfect efficiency & multiple dox conditions
* LV FACS practice from EA's single color transfections
* Passage
* Make new clear DMEM!

Wednesday August 1st:

* Confocal KB's transfections
* KB transfect eYFP
* NK induce DOX ladder
* LV transfect KD with Hef1A:TetR
* make red supplemented DMEM
* FACS LV's KD with reduced parameters, KB's two ALEXA:ROX expts (one with 15 pmol rep6 and 45 pmol of either input) (one with 30 pmol rep6 and 75 pmol of either input)

Thursday August 2nd:

* KB transfect in 35 mm dishes at 7:45 AM
* KB confocal with DM at 10:30 AM
* NK FACS DOX Ladder, efficiency
* NK transfect FF4 system, FF1 system
* Make new clear supplemented DMEM (Note, we only have one stock bottle left!)
* Split 4 plates of HEK293 at 1:6

Friday August 3rd:

* NK transfect :
* KB transfect 15,20,25,30 ALEXA:ROX
* LV transfect FF4 system?
* Split 4 plates of HEK293 at 1:6

Saturday August 4th:

* KB FACS with DM
* NK FACS

Week Nine: 8/6/12 - 8/12/12

Monday 8/6/12:

NK transfect DOX curve with 16 data points NK transfect FF4 system with varying amounts of decoy / miRFF4, and the Lac system KB transfect FF1 system Split 4 plates of HEK293s

Tuesday 8/7/12:

KB transfect 15,20,25,30 pmol of annealed reporter along with 3 positive controls - Transfection of Rep6+Alexa (July 5th) NK redo DOX transfection (with 16 data points?) NK induce DOX, IPTG Split 4 plates of HEK293s at 1:6 with Jenna Bring Giulio into TC - bring up a new vial of eYFPs and start passaging

Wednesday 8/8/12:

FACS KB FF1 and NK FF4 at 9:00 A.M. Supplemented red DMEM with Giulio FACS NK DOX Ladder and KB Alexa:ROX Jenna and Giulio split five plates of HEK293+eYFPs at 1:6 Eta retransfect FF4 Nathan induce DOX on yesterday's transfection KB retransfect ALEXA:ROX KB retransfect FF1 system (with TetRKrab this time) KB split four plates of HEK293s at 1:6

Thursday 8/9/12:

main culture underconfluent - only performed media change Eta and Nathan retransfected FF4 system FACS NK DOX ladder made supplemented clear media

Friday 8/10/12:

FACS KB A:R + 3X Inputs FACS KB FF1 Knockdown with TetR, TetR-Krab KB transfect A:R + U6:TetO inputs KB transfect FF1 with TetR, TetR-Krab and DOX in media NK transfect LacI? FF4, 16 data point DOX GA transfect constitutive colors Passage - 5 plates 1:6 for Sunday

Saturday 8/11/12:

Scope KB A:R with DNA inputs FACS EA FF4 NK induce DOX ladder Design meeting! Passage - 5 plates 1:6 for Monday

Week Eleven: 8/20/12 - 8/26/12

Monday 8/20/12:

Split 2 plates of 293s at 1:6 Split 1 plate of eYFPs at 1:6 Giulio split 293s 1:6

Wednesday 8/22/12:

Split 1 plate of eYPFs 1:12 for Saturday Split 1 plate of 293s 1:3 for Thursday Split 2 plates of 293s 1:12 for Saturday Seeded a 6 well plate for Jameel. One well of 1,000,000 293s and one well of 1,000,000 eYFPs Giulio split 293s Things to do: make media (thaw FBS!) bring up cells from frozen check if there is lab stock of FBS

Thursday 8/23/12:
giulio microinjection expt

Saturday 8/25/12:
made supplemented clear DMEM, supplemented red DMEM Split one plate of 293s 1:3, two plates 1:6 and one plate 1:12 Split one plate of eYPFs at 1:12 Thawing a bottle of FBS to aliquot

Sunday 8/26/12:
made 50 mL aliquots of FBS for iGEM split cells (on P20) 1:3, 1:6 and 1:12 transfected constitutive colors for imaging

Week Twelve: 8/27/12 - 9/2/12

Tuesday 8/28/12:

Split cells (on P21) 1:3, 1:6 and 1:12
seeded cells for Giulio for FACS training on Thursday

Wednesday 8/29/12:

Giulio : microinjection experiment with HeLa cells
image the constitutive color transfection
transfect A:R in triplicate
image A:R (2 - 9 PM)
FACS at 5 PM
make media
bring cells up from frozen stock
plate HeLa cells

Fall Tissue Culture Lab Notebook

9/20/12

NK transfect FF1 titration expt
Split 3 plates of 293s at 1:6

9/21/12

NK transfect FF1 titration expt
Split 3 plates of 293s at 1:6
GA and KB nucleofection of long reporter, long reporter + bulge, with either the correct or incorrrect input
KB transfect U6-TetO-S1 and U6-TetO-S6

9/22/12

KB transfect new longer RNA reporters and new inputs. FACS. Data looks good!
KB split two plates of 293s at 1:6 and one plate at 1:4. Supplemented clear media
NK FACS FF1 titration
NK transfect FF1 system

9/23/12

NK FACS FF1 Titration
NK split cells 1:6 into three plates

9/24/12
NK FACS FF1 Titration and retransfect FF1 system
NK split cells 1:6 into 3 plates

9/25/12

NK transfect FF1 system, HH_mKate and mKate_HH
NK Took out FBS to thaw to make more red media
NK Took out trypsin to thaw to replenish aliquots.
KB Split cells 1:6
9/26/12
NK made more red media
FAC FACS FF1 System
NK transfect FF1 System, HH_mKate and mKate_HH
NK Split cells 1:6 into three plates

9/27/12
NK FACS FF1 System, HH_mKate and mKate_HH
NK Split cells 1:6 into three plates
NK re-transfect FF1 System, HH_mKate and mKate_HH
9/28/12
NK FACS FF1 System, HH_mKate and mKate_HH.
NK Split cells 1:6 into three plates.

9/29/12
NK re-transfect HH_mKate, mKate_HH, Hef1a:mKate and Hef1a:mKate-int-FF4 as a back up.

10/1/12
NK FACS HH_mKate, mKate_HH, Hef1a:mKate and Hef1a:mKate-int-FF4.
Gave HEK293 Cells to Giulio. Will refresh working cultures tomorrow.

10/3/12
NK- brought up new cells from the -140.

Retrieved from "http://2012.igem.org/Team:MIT/Notebook"