Team:MIT/Promoter Group Notebook

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Promoter Group Notebook

6/6/16:
ARB KB SC TM
Made LB Broth

6/7/16:
ARB KB SC TM
Screwed up yesterday, made more LB Broth
Made 50 mL overnight culture of hef1a-tagBFP

6/8/16:
ARB KB SC TM
Midiprepped hef1a-tagBFP; midiprep failed (very low concentration)

6/9/16
ARB KB SC TM
Midiprep for hef1a-tagBFP; failed again

• One lysate solution of the two spilled a bit during vacuuming, will probably use the other for transfection if it results in higher concentration
  
  

6/16/16
ARB KB SC TM
Midiprep for hef1a - tagBFP failed: yielded concentration of 12.7 for the DNA

6/17/16
ARB KB SC TM
Got new hef1a- tagBFP from Nelson
6/20/16
ARB KB SC TM
Transformed Nelson’s hef1a- tagBFP
Split HEK cells and seeded a 24 well plate for transfection optimization experiment

6/22/16
KB SC ARB TM
Golden gate reaction for pERE3
Transformation for pERE3
Pouring LB Agar plates→ failed
Picked colonies and grew overnight cultures for Nelson’s hef1a-tagBFP

6/23/16
KB SC ARB
LB Agar plates poured with Kan and XGal
ER alpha arrived from DNASU → kept in -80
Picked colonies and grew overnight cultures for pERE3

6/24/16
KB ARB SC
Miniprep pERE3

6/27/16
KB SC ARB
Golden gate, transformation, and plating for pERE5 & 6
Restriction digest and gel for pERE3
Retrieved MCF7 cells, put in the -144

6/28/16
KB SC ARB
Sent in pERE3 for sequencing
Thawed and propogated MCF-7 cells
Picked colonies for ERE5 and ERE6

6/29/16
KB SC ARB
Miniprep for ERE5 & 6
Restriction digest for ERE5 & 6
Ran gel for ERE5
LR rxn for hef1a - ER alpha
Transformation & plating of hef1a - ER alpha

6/30/16
KB SC ARB
Cell splitting
Start MCF7 cell line
Ran gel for ERE6
Imaged gels for ERE5 & 6
Pick colonies for hef1a/ER alpha
LR rxn & transformation for pERE_3/eYFP
Sent in ERE_5/6 for sequencing
Split HEK cells

7/1/16
KB SC ARB
Picked colonies and grew 5 mL cultures for pERE3-eYFP
LR Rxn to make pERE5 & 6 - eYFP

7/2/16
AR SC
Transformed and plated LRs for pERE5 & 6 - eYFP
Miniprepped pERE3-eYFP

7/5/16
KB SC ARB
Sequencing and gel for pERE3-eYFP

7/6/16
KB SC ARB
Sent for sequencing hef1a-ER alpha; worked on cytoflow; grew colonies overnight; cleaned out freezer box and labeled some sequenced verified stuff
pERE_6-mEYFP tube 3 or 4 (likely 4)
Split HEK cells

7/7/16
ARB KB SC
Split HEK cells
Midiprep for pERE_3-mEYFP and made glycerol stock
Sequence verify ER alpha pENTR
Miniprep for pERE_5/6-mEYFP and hef1a-ERalpha
Digest, Image, and Gel pERE5/6-mEYFP and hef1a-ERalpha

7/8/16
KB SC ARB
Midiprepped pERE5 & 6 -meYFP → failed
Re transformed the sequenced verified DNA of pERE5 & 6 - meYFP

7/9/16
ARB KB
Picked colonies from transformation and grew up 5 mL overnight cultures

7/10/16
ARB KB
Made 50 mL cultures of pERE5 & 6 - eYFP and incubated for 4 hours for midiprep
Screwed up the cultures → did not midiprep
Made new 5 mL cultures
LR Reaction for ERE - Rep

7/11/16
KB SC ARB
Incubated 50 mL cultures with amp for midiprep
Made dilutions of E2
Midiprepped pERE5 & 6 - eYFP
Transformed LRs from yesterday

7/12/16
KB SC ARB
Picked colonies and grew 5 mL cultures for pERE-Rep

7/13/16
KB SC ARB
Miniprepped pERE-Rep
Digest, Gel, Sequence

7/14/16
KB SC ARB
Split HEK cells
Grew overnight cultures for pERE-Rep

7/15/16:
KB SC ARB
Midiprepped pERE-Rep

7/18/16:
KB SC ARB
Sequenced pERE-Rep cultures

7/19/16:
KB SC ARB
LR for pTFF1-eYFP

7/20/16:
KB SC ARB
LR reaction for pRep - Recombinase
Transformed pTFF1-eYFP

7/19/16:
KB SC ARB
Transformed pRep - Recombinase LRs

7/20/16:
KB SC ARB
Flow cytometry prep
MCF-7 Transfection Optimization Flow Cytometry
Picked colonies and grew 5 mL cultures

7/21/16:
ARB KB SC
Miniprepped pRep-Recombinase constructs

7/22/16:
ARB KB SC
Split HEK293 and MCF7
Seeded 12 well plate for HEK
Seeded 24 well plate for MCF7 for expt. 0 ((MCF7 → coarse induction of pERE3/5/6 - eYFP; varying time and conc.)
Digested, ran gel for pRepressor - recombinase constructs (failed → determined we didn’t dilute pENTRYs)
Sequenced pTFF1 - eYFP

7/23/16:
ARB KB SC
Transfected MCF7 with pERE3/5/6 for expt. 0
Induced with estrogen at 3 and 10 hrs. post-transfection
7/24/16:
ARB KB SC
27 hr. post-transfection 10 nM estrogen induction

7/25/16:
ARB KB SC
Split ISH, tHESC, and MCF7
Made frozen stocks of ISH and tHESC
Seeded a well plate with MCF7 for expt. 1(MCF7 → fine induction of pERE3/5 - eYFP)
Flow cytometry for expt. 0 in MCF7
Data analysis of flow cytometry
Thawed and made new plate of HEK293

7/26/16:
ARB KB SC
Resuspended pHybrid from IDT
Diluted estrogen to necessary concentrations for expt. 1
Transfected MCF-7 with pERE_3/5 - eYFP [expt. 1]
LR for pRepressor - Recombinase constructs
Presented lab meeting

7/27/16:
ARB KB SC
Golden gate to make pHybrid into an entry vector
Transformation of pRepressor - Recombinase constructs
Transformation of golden gate for pHybrid pENTRY
LR reactions for pERE_x - TAL21
Induced expt. 1 with various estrogen dilutions (nM)

7/28/16:
ARB KB SC
Flow cytometry for expt. 1 (failed --> low cell density, 1% transfection efficiency)
Split MCF7 and seeded well plate to repeat expt. 1 for pERE3 - eYFP fine induction
Split tHESC and ISH and seeded well plate for optimization expt.
Grow overnight colonies of pRepressor - Recombinase constructs
Transformation of pERE_x - TAL21 constructs

7/29/16:
ARB KB SC
Miniprepped pEREx Recombinase constructs
Digest of pEREx Recombinase constructs
Goldengate for several pPRE constructs
Sequence ERalpha entry vector

7/30/2016
ARB KB
Transfected MCF7, ISH, tHESC (repeat expt.1 /optimization expt)

7/31/2016
ARB
Took images of cells - tHESC no transfection

8/1/16:
ARB KB SC
Cytometery for expt. 1 redo (E3 in MCF7) and analysis in flowcyto
Golden gate redo of pPRE3/pHybrid.
Transformation of golden gate.
Grew overnights of pERE5 -repressor constructs
Split ISH, MCF7 and seeded well plates.

8/2/16:
KB SC ARB TM
Transfection of expt. 1 for E3/E5 in ISH and expt. 1 for E5 in MCF7
Mini prep for pERE5 - repressor constructs; ran digest, gel, and sequencing as well
Re-transformation of pDonor for transfections.
Set up overnight cultures for pRepressor - recombinase
Induced transfection with estrogen amounts

8/3/16:
KB ARB SC
Midiprep of pRepressor - recombinase
Made media for tHESC/ISH/MCF7
Split tHESC and seeded a well plate for optimization re-do
Diluted and sterilized Kan stocks
Grow overnights for pENTR pPRE3/pHybrid

8/4/16:
KB ARB SC
Flow cytometry preparation
Flow cytometry (BT)
Miniprep for PRE3 and hybrid promoter
Grew up cultures and midiprepped pDONR-GG
Digest, gel, and sequencing for PRE & hybrid
Split and seeded plates for MCF7 and ISH
Transfection for tHESC

8/5/16:
KB ARB SC
Transfected with expt. 2 DNA in MCF7 and ISH
Sent in pPRE3 and pHybrid entry vectors for sequencing (previous attempt priming failed)
K-killed LR reactions for pPRE3/pHybrid -eYFP
Changed media on tHESC cells

8/6/16
KB
Induced wells with E2

8/7/16
ARB SC
Flow cytometry prep
pPRE3 + pHybrid eYFP transformation

8/8/16
KB ARB SC
Split ISH and seeded a 24 well plate
Picked colonies and grew up 5 mL overnight cultures for pPRE3 & pHybrid

8/9/16
KB ARB SC
Miniprepped progesterone responsive promoter
Sent pPRE in for sequencing
Picked colonies and grew up new cultures for hybrid promoter
Transfection into ISH

8/10/16
KB ARB SC
Attempted to dissolve MPA in ethanol
Miniprepped hybrid promoter
Sent in pHybrid for sequencing

8/11/16
KB ARB SC
Grew up midiprep 50 mL cultures
Flow cytometry prep
Midiprep
Grew new 50 mL cultures

8/12/16
KB ARB SC
Flow cytometry prep
Golden gate
Transformation
Midiprep
Split MCF7 and made new media

8/27/16
SC
Split MCF7 and seeded a 17 well plate

8/28/16
SC
Transfection of MCF7 with pERE-Repressor

8/29/16
SC
Estrogen induction for transfected cells

8/30/16
SC
Flow cytometry preparation
Cytometry

9/4/16
KB
Split and seeded MCF7

9/5/16
ARB
Transfected MCF7 with pERExN constructs

9/6/16
ARB KB
Induced MCF7 pERExn with micromolar induction (vehicle, .5 uM, … 10 uM)
Grew overnights for midi preps

9/8/16
SC ARB
Pelleted bacterial colonies in order to prepare for midiprep
Changed media on MCF7 and tried to salvage poor cell density
LR of pP4 eYFP, pE2 mk8

9/9/16
SC KB ARB
Midipreps of several different constructs in order to prepare for tHESC electroporation

9/10/16
KB ARB
Electroporation of tHESC

9/11/16
KB ARB
Induction with E2 and P4 (note vehicle for E2 was 100% ethanol and for P4 was DMEM/F12)

9/12/16
ARB SC KB
Cytometry of tHESC induced samples

9/13/16
KB
Data analysis for cytometry experiment.

9/14/16
KB
Split MCF7 and seeded a well plate.

9/15/16
SC
Transfected MCF7 with promoter-repressor experiment

9/16/16
KB
Induced wells with estrogen.

9/17/16
ARB
Cytometry for the MCF7 repressor cascade experiment.
Split tHESC and seeded a well plate.

9/18/16
ARB
Electroporation of tHESC with pP4xeYFP, pHybrid constructs

9/19/16
ARB
Induction of estrogen and progesterone on tHESC experiment

9/20/16
ARB KB
Flow cytometry of tHESC experiment
Data analysis of results

9/21/16
ARB
Split MCF7 and seeded a well plate.

9/22/16
ARB
Electroporation of tHESC to test pERE3 with vehicle control.

9/23/16
ARB
Induction of pERE3 with estrogen.

9/24/16
ARB KB
Cytometry of tHESC vehicle test experiment for pERE3

9/25/16
KB
Data analysis for tHESC experiment.
Split MCF7 and seeded a well plate.
Set up overnight colonies of hef1a-mKate for midiprepping.

9/26/16
SC
Took out and spun down the midiprep colonies.
Transfected MCF7 with promoter-repressor experiment re-try

9/27/16
KB
Induced cells with estrogen.

9/28/16
KB ARB
Flow cytometry for MCF7 experiment --failed due to low cell density and bad tfxn efficiency.

9/29/16
ARB
Split MCF7 cells with advice from Christi Cook on aseptic technique.

9/30/16
KB
Data analysis for recombinase group -- bleed through correction.

10/6/16
KB ARB
Split MCF7 and seeded a well plate.

10/7/16
KB ARB
Transfected MCF7 with final promoter experiment - PRE promoters, Hybrid
Electroporation of tHESC with final promoter experiment - PRE promoters fine induction, hybrid

10/8/16
KB
Induced wells with estrogen and progesterone.

10/9/16
KB ARB
Imaged cells using the Zeiss
Flow cytometry for tHESC and MCF7 experiment
Data analysis of results