Difference between revisions of "Team:MIT/Promoter Group Notebook"
(Created page with "<h1 style="color:#f20253; text-align: center; font-size: 40px; line-height: 40px;">Notebook Group Notebook</h1> <p> 6/6/16: ARB KB SC TM Made LB Broth 6/7/16: ARB KB SC TM...") |
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| − | 6/6/16: | + | 6/6/16: <br> |
| − | ARB KB SC TM | + | ARB KB SC TM <br> |
| − | Made LB Broth | + | Made LB Broth <br> <br> |
| − | 6/7/16: | + | 6/7/16: <br> |
| − | ARB KB SC TM | + | ARB KB SC TM <br> |
| − | Screwed up yesterday, made more LB Broth | + | Screwed up yesterday, made more LB Broth <br> |
| − | Made 50 mL overnight culture of hef1a-tagBFP | + | Made 50 mL overnight culture of hef1a-tagBFP <br> <br> |
| − | 6/8/16: | + | 6/8/16: <br> |
| − | ARB KB SC TM | + | ARB KB SC TM <br> |
| − | Midiprepped hef1a-tagBFP; midiprep failed (very low concentration) | + | Midiprepped hef1a-tagBFP; midiprep failed (very low concentration) <br> <br> |
| − | 6/9/16 | + | 6/9/16 <br> |
| − | ARB KB SC TM | + | ARB KB SC TM <br> |
| − | Midiprep for hef1a-tagBFP; failed again | + | Midiprep for hef1a-tagBFP; failed again <br> |
| − | *One lysate solution of the two spilled a bit during vacuuming, will probably use the other for transfection if it results in higher concentration | + | *One lysate solution of the two spilled a bit during vacuuming, will probably use the other for transfection if it results in higher concentration <br> <br> |
| − | 6/16/16 | + | 6/16/16 <br> |
| − | ARB KB SC TM | + | ARB KB SC TM <br> |
| − | Midiprep for hef1a - tagBFP failed: yielded concentration of 12.7 for the DNA | + | Midiprep for hef1a - tagBFP failed: yielded concentration of 12.7 for the DNA <br> <br> |
| − | 6/17/16 | + | 6/17/16 <br> |
| − | ARB KB SC TM | + | ARB KB SC TM <br> |
| − | Got new hef1a- tagBFP from Nelson | + | Got new hef1a- tagBFP from Nelson <br> |
| − | 6/20/16 | + | 6/20/16 <br> |
| − | ARB KB SC TM | + | ARB KB SC TM <br> |
| − | Transformed Nelson’s hef1a- tagBFP | + | Transformed Nelson’s hef1a- tagBFP <br> |
| − | Split HEK cells and seeded a 24 well plate for transfection optimization experiment | + | Split HEK cells and seeded a 24 well plate for transfection optimization experiment <br> <br> |
| − | 6/22/16 | + | 6/22/16 <br> |
| − | Golden gate reaction for pERE3 | + | KB SC ARB TM <br> |
| − | Transformation for pERE3 | + | Golden gate reaction for pERE3 <br> |
| − | Pouring LB Agar plates→ failed | + | Transformation for pERE3 <br> |
| − | Picked colonies and grew overnight cultures for Nelson’s hef1a-tagBFP | + | Pouring LB Agar plates→ failed <br> |
| + | Picked colonies and grew overnight cultures for Nelson’s hef1a-tagBFP <br> <br> | ||
| − | 6/23/16 | + | 6/23/16 <br> |
| − | LB Agar plates poured with Kan and XGal | + | KB SC ARB <br> |
| − | ER alpha arrived from DNASU → kept in -80 | + | LB Agar plates poured with Kan and XGal <br> |
| − | Picked colonies and grew overnight cultures for pERE3 | + | ER alpha arrived from DNASU → kept in -80 <br> |
| + | Picked colonies and grew overnight cultures for pERE3 <br> <br> | ||
| − | 6/24/16 | + | 6/24/16 <br> |
| − | Miniprep pERE3 | + | Miniprep pERE3 <br> <br> |
| − | 6/27/16 | + | 6/27/16 <br> |
| − | Golden gate, transformation, and plating for pERE5 & 6 | + | KB SC ARB <br> |
| − | Restriction digest and gel for pERE3 | + | Golden gate, transformation, and plating for pERE5 & 6 <br> |
| − | Retrieved MCF7 cells, put in the -144 | + | Restriction digest and gel for pERE3 <br> |
| + | Retrieved MCF7 cells, put in the -144 <br> <br> | ||
| − | 6/28/16 | + | 6/28/16 <br> |
| − | Sent in pERE3 for sequencing | + | KB SC ARB <br> |
| − | Thawed and propogated MCF-7 cells | + | Sent in pERE3 for sequencing <br> |
| − | Picked colonies for ERE5 and ERE6 | + | Thawed and propogated MCF-7 cells <br> |
| + | Picked colonies for ERE5 and ERE6 <br> | ||
| + | <br> | ||
| + | 6/29/16 <br> | ||
| + | KB SC ARB <br> | ||
| + | Miniprep for ERE5 & 6 <br> | ||
| + | Restriction digest for ERE5 & 6 <br> | ||
| + | Ran gel for ERE5 <br> | ||
| + | LR rxn for hef1a - ER alpha <br> | ||
| + | Transformation & plating of hef1a - ER alpha <br> <br> | ||
| − | 6/ | + | 6/30/16 <br> |
| − | + | KB SC ARB <br> | |
| − | + | Cell splitting <br> | |
| − | + | Start MCF7 cell line <br> | |
| − | + | Ran gel for ERE6 <br> | |
| − | + | Imaged gels for ERE5 & 6 <br> | |
| + | Pick colonies for hef1a/ER alpha <br> | ||
| + | LR rxn & transformation for pERE_3/eYFP <br> | ||
| + | Sent in ERE_5/6 for sequencing <br> | ||
| + | Split HEK cells <br> <br> | ||
| − | + | 7/1/16 <br> | |
| − | + | KB SC ARB <br> | |
| − | + | Picked colonies and grew 5 mL cultures for pERE3-eYFP <br> | |
| − | + | LR Rxn to make pERE5 & 6 - eYFP <br> <br> | |
| − | + | ||
| − | + | ||
| − | LR | + | |
| − | + | ||
| − | + | ||
| − | 7/ | + | 7/2/16 <br> |
| − | + | AR SC <br> | |
| − | + | Transformed and plated LRs for pERE5 & 6 - eYFP <br> | |
| + | Miniprepped pERE3-eYFP <br> <br> | ||
| − | 7/ | + | 7/5/16 <br> |
| − | + | KB SC ARB <br> | |
| − | + | Sequencing and gel for pERE3-eYFP <br> <br> | |
| − | + | ||
| − | |||
| − | |||
| + | 7/6/16 <br> | ||
| + | Sent for sequencing hef1a-ER alpha; worked on cytoflow; grew colonies overnight; cleaned out freezer box and labeled some sequenced verified stuff <br> | ||
| + | pERE_6-mEYFP tube 3 or 4 (likely 4) <br> | ||
| + | Split HEK cells <br> <br> | ||
| − | 7/ | + | 7/7/16 <br> |
| − | + | ARB KB SC <br> | |
| − | + | Split HEK cells <br> | |
| − | + | Midiprep for pERE_3-mEYFP and made glycerol stock <br> | |
| + | Sequence verify ER alpha pENTR <br> | ||
| + | Miniprep for pERE_5/6-mEYFP and hef1a-ERalpha <br> | ||
| + | Digest, Image, and Gel pERE5/6-mEYFP and hef1a-ERalpha <br> <br> | ||
| − | 7/ | + | 7/8/16 <br> |
| − | + | KB SC ARB <br> | |
| − | + | Midiprepped pERE5 & 6 -meYFP → failed <br> | |
| − | + | Re transformed the sequenced verified DNA of pERE5 & 6 - meYFP <br> <br> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | 7/ | + | 7/9/16 <br> |
| − | + | ARB KB <br> | |
| − | + | Picked colonies from transformation and grew up 5 mL overnight cultures <br> <br> | |
| − | 7/ | + | 7/10/16 <br> |
| − | ARB KB | + | ARB KB <br> |
| − | + | Made 50 mL cultures of pERE5 & 6 - eYFP and incubated for 4 hours for midiprep <br> | |
| + | Screwed up the cultures → did not midiprep <br> | ||
| + | Made new 5 mL cultures <br> | ||
| + | LR Reaction for ERE - Rep <br> <br> | ||
| − | 7/ | + | 7/11/16 <br> |
| − | + | KB SC ARB <br> | |
| − | + | Incubated 50 mL cultures with amp for midiprep <br> | |
| − | + | Made dilutions of E2 <br> | |
| − | + | Midiprepped pERE5 & 6 - eYFP <br> | |
| − | + | Transformed LRs from yesterday <br> <br> | |
| − | 7/ | + | 7/12/16 <br> |
| − | + | KB SC ARB <br> | |
| − | + | Picked colonies and grew 5 mL cultures for pERE-Rep <br> <br> | |
| − | + | ||
| − | + | ||
| − | 7/ | + | 7/13/16 <br> |
| − | + | KB SC ARB <br> | |
| + | Miniprepped pERE-Rep <br> | ||
| + | Digest, Gel, Sequence <br> <br> | ||
| − | 7/ | + | 7/14/16 <br> |
| − | + | KB SC ARB <br> | |
| − | + | Split HEK cells <br> | |
| + | Grew overnight cultures for pERE-Rep <br> <br> | ||
| − | 7/ | + | 7/15/16: <br> |
| − | + | KB SC ARB <br> | |
| − | + | Midiprepped pERE-Rep <br> <br> | |
| − | 7/ | + | 7/18/16: <br> |
| − | + | KB SC ARB <br> | |
| − | + | ||
| − | + | ||
Sequenced pERE-Rep cultures | Sequenced pERE-Rep cultures | ||
| + | <br> <br> | ||
| + | 7/19/16: <br> | ||
| + | KB SC ARB <br> | ||
| + | LR for pTFF1-eYFP <br> <br> | ||
| − | 7/ | + | 7/20/16: <br> |
| − | LR for pTFF1-eYFP | + | KB SC ARB <br> |
| + | LR reaction for pRep - Recombinase <br> | ||
| + | Transformed pTFF1-eYFP <br> <br> | ||
| − | 7/ | + | 7/19/16: <br> |
| − | + | KB SC ARB <br> | |
| − | + | Transformed pRep - Recombinase LRs <br> <br> | |
| − | 7/ | + | 7/20/16: <br> |
| − | + | KB SC ARB <br> | |
| + | Flow cytometry prep <br> | ||
| + | MCF-7 Transfection Optimization Flow Cytometry <br> | ||
| + | Picked colonies and grew 5 mL cultures <br> <br> | ||
| − | + | 7/21/16: <br> | |
| − | + | ARB KB SC <br> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | 7/21/16: | + | |
| − | ARB KB SC | + | |
Miniprepped pRep-Recombinase constructs | Miniprepped pRep-Recombinase constructs | ||
| + | <br> <br> | ||
| + | 7/22/16: <br> | ||
| + | ARB KB SC <br> | ||
| + | Split HEK293 and MCF7 <br> | ||
| + | Seeded 12 well plate for HEK <br> | ||
| + | Seeded 24 well plate for MCF7 for expt. 0 ((MCF7 → coarse induction of pERE3/5/6 - eYFP; varying time and conc.) <br> | ||
| + | Digested, ran gel for pRepressor - recombinase constructs (failed → determined we didn’t dilute pENTRYs) <br> | ||
| + | Sequenced pTFF1 - eYFP <br> <br> | ||
| + | |||
| + | 7/23/16: <br> | ||
| + | ARB KB SC <br> | ||
| + | Transfected MCF7 with pERE3/5/6 for expt. 0 <br> | ||
| + | Induced with estrogen at 3 and 10 hrs. post-transfection <br> | ||
| − | + | 7/24/16: <br> | |
| − | + | ARB KB SC <br> | |
| − | + | 27 hr. post-transfection 10 nM estrogen induction <br> <br> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | 7/24/16: | + | |
| − | ARB KB SC | + | |
| − | 27 hr. post-transfection 10 nM estrogen induction | + | |
| − | 7/25/16: | + | 7/25/16: <br> |
| − | ARB KB SC | + | ARB KB SC <br> |
| − | Split ISH, tHESC, and MCF7 | + | Split ISH, tHESC, and MCF7 <br> |
| − | Made frozen stocks of ISH and tHESC | + | Made frozen stocks of ISH and tHESC <br> |
| − | Seeded a well plate with MCF7 for expt. 1(MCF7 → fine induction of pERE3/5 - eYFP) | + | Seeded a well plate with MCF7 for expt. 1(MCF7 → fine induction of pERE3/5 - eYFP) <br> |
| − | Flow cytometry for expt. 0 in MCF7 | + | Flow cytometry for expt. 0 in MCF7 <br> |
| − | Data analysis of flow cytometry | + | Data analysis of flow cytometry <br> |
| − | Thawed and made new plate of HEK293 | + | Thawed and made new plate of HEK293 <br> <br> |
| − | 7/26/16: | + | 7/26/16: <br> |
| − | ARB KB SC | + | ARB KB SC <br> |
| − | Resuspended pHybrid from IDT | + | Resuspended pHybrid from IDT <br> |
| − | Diluted estrogen to necessary concentrations for expt. 1 | + | Diluted estrogen to necessary concentrations for expt. 1 <br> |
| − | Transfected MCF-7 with pERE_3/5 - eYFP [expt. 1] | + | Transfected MCF-7 with pERE_3/5 - eYFP [expt. 1] <br> |
| − | LR for pRepressor - Recombinase constructs | + | LR for pRepressor - Recombinase constructs <br> |
| − | Presented lab meeting | + | Presented lab meeting <br> <br> |
| − | 7/27/16: | + | 7/27/16: <br> |
| − | ARB KB SC | + | ARB KB SC <br> |
| − | Golden gate to make pHybrid into an entry vector | + | Golden gate to make pHybrid into an entry vector <br> |
| − | Transformation of pRepressor - Recombinase constructs | + | Transformation of pRepressor - Recombinase constructs <br> |
| − | Transformation of golden gate for pHybrid pENTRY | + | Transformation of golden gate for pHybrid pENTRY <br> |
| − | LR reactions for pERE_x - TAL21 | + | LR reactions for pERE_x - TAL21 <br> |
| − | Induced expt. 1 with various estrogen dilutions (nM) | + | Induced expt. 1 with various estrogen dilutions (nM) <br> <br> |
| − | 7/28/16: | + | 7/28/16: <br> |
| − | ARB KB SC | + | ARB KB SC <br> |
| − | Flow cytometry for expt. 1 (failed --> low cell density, 1% transfection efficiency) | + | Flow cytometry for expt. 1 (failed --> low cell density, 1% transfection efficiency) <br> |
| − | Split MCF7 and seeded well plate to repeat expt. 1 for pERE3 - eYFP fine induction | + | Split MCF7 and seeded well plate to repeat expt. 1 for pERE3 - eYFP fine induction <br> |
| − | Split tHESC and ISH and seeded well plate for optimization expt. | + | Split tHESC and ISH and seeded well plate for optimization expt. <br> |
| − | Grow overnight colonies of pRepressor - Recombinase constructs | + | Grow overnight colonies of pRepressor - Recombinase constructs <br> |
| − | Transformation of pERE_x - TAL21 constructs | + | Transformation of pERE_x - TAL21 constructs <br> <br> |
| − | 7/29/16: | + | 7/29/16: <br> |
| − | ARB KB SC | + | ARB KB SC <br> |
| − | Miniprepped pEREx Recombinase constructs | + | Miniprepped pEREx Recombinase constructs <br> |
| − | Digest of pEREx Recombinase constructs | + | Digest of pEREx Recombinase constructs <br> |
| − | Goldengate for several pPRE constructs | + | Goldengate for several pPRE constructs <br> |
| − | Sequence ERalpha entry vector | + | Sequence ERalpha entry vector <br> <br> |
| − | 7/30/2016 | + | 7/30/2016 <br> |
| − | ARB KB | + | ARB KB <br> |
| − | Transfected MCF7, ISH, tHESC (repeat expt.1 /optimization expt) | + | Transfected MCF7, ISH, tHESC (repeat expt.1 /optimization expt) <br> <br> |
| − | 7/31/2016 | + | 7/31/2016 <br> |
| − | ARB | + | ARB <br> |
| − | Took images of cells - tHESC no transfection | + | Took images of cells - tHESC no transfection <br> <br> |
| − | 8/1/16: | + | 8/1/16: <br> |
| − | ARB KB SC | + | ARB KB SC <br> |
| − | Cytometery for expt. 1 redo (E3 in MCF7) and analysis in flowcyto | + | Cytometery for expt. 1 redo (E3 in MCF7) and analysis in flowcyto <br> |
| − | Golden gate redo of pPRE3/pHybrid. | + | Golden gate redo of pPRE3/pHybrid. <br> |
| − | Transformation of golden gate. | + | Transformation of golden gate. <br> |
| − | Grew overnights of pERE5 -repressor constructs | + | Grew overnights of pERE5 -repressor constructs <br> |
| − | Split ISH, MCF7 and seeded well plates. | + | Split ISH, MCF7 and seeded well plates. <br> <br> |
| − | 8/2/16: | + | 8/2/16: <br> |
| − | KB SC ARB TM | + | KB SC ARB TM <br> |
| − | Transfection of expt. 1 for E3/E5 in ISH and expt. 1 for E5 in MCF7 | + | Transfection of expt. 1 for E3/E5 in ISH and expt. 1 for E5 in MCF7 <br> |
| − | Mini prep for pERE5 - repressor constructs; ran digest, gel, and sequencing as well | + | Mini prep for pERE5 - repressor constructs; ran digest, gel, and sequencing as well <br> |
| − | Re-transformation of pDonor for transfections. | + | Re-transformation of pDonor for transfections. <br> |
| − | Set up overnight cultures for pRepressor - recombinase | + | Set up overnight cultures for pRepressor - recombinase <br> |
| − | Induced transfection with estrogen amounts | + | Induced transfection with estrogen amounts <br> <br> |
| − | 8/3/16: | + | 8/3/16: <br> |
| − | KB ARB SC | + | KB ARB SC <br> |
| − | Midiprep of pRepressor - recombinase | + | Midiprep of pRepressor - recombinase <br> |
| − | Made media for tHESC/ISH/MCF7 | + | Made media for tHESC/ISH/MCF7 <br> |
| − | Split tHESC and seeded a well plate for optimization re-do | + | Split tHESC and seeded a well plate for optimization re-do <br> |
| − | Diluted and sterilized Kan stocks | + | Diluted and sterilized Kan stocks <br> |
| − | Grow overnights for pENTR pPRE3/pHybrid | + | Grow overnights for pENTR pPRE3/pHybrid <br> <br> |
| − | 8/4/16: | + | 8/4/16: <br> |
| − | KB ARB SC | + | KB ARB SC <br> |
| − | Flow cytometry preparation | + | Flow cytometry preparation <br> |
| − | Flow cytometry (BT) | + | Flow cytometry (BT) <br> |
| − | Miniprep for PRE3 and hybrid promoter | + | Miniprep for PRE3 and hybrid promoter <br> |
| − | Grew up cultures and midiprepped pDONR-GG | + | Grew up cultures and midiprepped pDONR-GG <br> |
| − | Digest, gel, and sequencing for PRE & hybrid | + | Digest, gel, and sequencing for PRE & hybrid <br> |
| − | Split and seeded plates for MCF7 and ISH | + | Split and seeded plates for MCF7 and ISH <br> |
| − | Transfection for tHESC | + | Transfection for tHESC <br> <br> |
| − | 8/5/16: | + | 8/5/16: <br> |
| − | KB ARB SC | + | KB ARB SC <br> |
| − | Transfected with expt. 2 DNA in MCF7 and ISH | + | Transfected with expt. 2 DNA in MCF7 and ISH <br> |
| − | Sent in pPRE3 and pHybrid entry vectors for sequencing (previous attempt priming failed) | + | Sent in pPRE3 and pHybrid entry vectors for sequencing (previous attempt priming failed) <br> |
| − | K-killed LR reactions for pPRE3/pHybrid -eYFP | + | K-killed LR reactions for pPRE3/pHybrid -eYFP <br> |
| − | Changed media on tHESC cells | + | Changed media on tHESC cells <br> <br> |
| − | 8/6/16 | + | 8/6/16 <br> |
| − | KB | + | KB <br> |
| − | Induced wells with E2 | + | Induced wells with E2 <br> |
| − | 8/7/16 | + | 8/7/16 <br> |
| − | ARB SC | + | ARB SC <br> |
| − | Flow cytometry prep | + | Flow cytometry prep <br> |
| − | pPRE3 + pHybrid eYFP transformation | + | pPRE3 + pHybrid eYFP transformation <br> <br> |
| − | 8/8/16 | + | 8/8/16 <br> |
| − | KB ARB SC | + | KB ARB SC <br> |
| − | Split ISH and seeded a 24 well plate | + | Split ISH and seeded a 24 well plate <br> |
| − | Picked colonies and grew up 5 mL overnight cultures for pPRE3 & pHybrid | + | Picked colonies and grew up 5 mL overnight cultures for pPRE3 & pHybrid <br> <br> |
| − | 8/9/16 | + | 8/9/16 <br> |
| − | KB ARB SC | + | KB ARB SC <br> |
| − | Miniprepped progesterone responsive promoter | + | Miniprepped progesterone responsive promoter <br> |
| − | Sent pPRE in for sequencing | + | Sent pPRE in for sequencing <br> |
| − | Picked colonies and grew up new cultures for hybrid promoter | + | Picked colonies and grew up new cultures for hybrid promoter <br> |
| − | Transfection into ISH | + | Transfection into ISH <br> <br> |
| − | 8/10/16 | + | 8/10/16 <br> |
| − | KB ARB SC | + | KB ARB SC <br> |
| − | Attempted to dissolve MPA in ethanol | + | Attempted to dissolve MPA in ethanol <br> |
| − | Miniprepped hybrid promoter | + | Miniprepped hybrid promoter <br> |
| − | Sent in pHybrid for sequencing | + | Sent in pHybrid for sequencing <br> <br> |
| − | 8/11/16 | + | 8/11/16 <br> |
| − | KB ARB SC | + | KB ARB SC <br> |
| − | Grew up midiprep 50 mL cultures | + | Grew up midiprep 50 mL cultures <br> |
| − | Flow cytometry prep | + | Flow cytometry prep <br> |
| − | + | Midiprep <br> | |
| − | Midiprep | + | Grew new 50 mL cultures <br> <br> |
| − | Grew new 50 mL cultures | + | |
| − | 8/12/16 | + | 8/12/16 <br> |
| − | KB ARB SC | + | KB ARB SC <br> |
| − | Flow cytometry prep | + | Flow cytometry prep <br> |
| − | Golden gate | + | Golden gate <br> |
| − | Transformation | + | Transformation <br> |
| − | Midiprep | + | Midiprep <br> |
| − | Split MCF7 and made new media | + | Split MCF7 and made new media <br> <br> |
| − | 8/27/16 | + | 8/27/16 <br> |
| − | SC | + | SC <br> |
| − | Split MCF7 and seeded a 17 well plate | + | Split MCF7 and seeded a 17 well plate <br> <br> |
| − | 8/28/16 | + | 8/28/16 <br> |
| − | SC | + | SC <br> |
| − | Transfection of MCF7 | + | Transfection of MCF7 with pERE-Repressor <br> <br> |
| − | 8/29/16 | + | 8/29/16 <br> |
| − | SC | + | SC <br> |
| − | Estrogen induction for transfected cells | + | Estrogen induction for transfected cells <br> <br> |
| − | 8/30/16 | + | 8/30/16 <br> |
| − | SC | + | SC <br> |
| − | Flow cytometry preparation | + | Flow cytometry preparation <br> |
| − | Cytometry | + | Cytometry <br> <br> |
| − | 9/4/16 | + | 9/4/16 <br> |
| − | KB | + | KB <br> |
| − | Split and seeded MCF7 | + | Split and seeded MCF7 <br> <br> |
| − | 9/5/16 | + | 9/5/16 <br> |
| − | ARB | + | ARB <br> |
| − | Transfected MCF7 with pERExN constructs | + | Transfected MCF7 with pERExN constructs <br> <br> |
| − | 9/6/16 | + | 9/6/16 <br> |
| − | ARB | + | ARB KB<br> |
| − | Induced MCF7 pERExn with micromolar induction (vehicle, .5 uM, … 10 uM) | + | Induced MCF7 pERExn with micromolar induction (vehicle, .5 uM, … 10 uM) <br> |
| − | + | Grew overnights for midi preps <br> <br> | |
| − | Grew overnights for midi preps | + | |
| − | 9/8/16 | + | 9/8/16 <br> |
| − | SC ARB | + | SC ARB <br> |
| − | Pelleted bacterial colonies in order to prepare for midiprep | + | Pelleted bacterial colonies in order to prepare for midiprep <br> |
| − | Changed media on MCF7 and tried to salvage poor cell density | + | Changed media on MCF7 and tried to salvage poor cell density <br> |
| − | LR of pP4 eYFP, pE2 mk8 | + | LR of pP4 eYFP, pE2 mk8 <br> <br> |
| − | 9/9/16 | + | 9/9/16 <br> |
| − | SC KB ARB | + | SC KB ARB <br> |
| − | Midipreps of several different constructs in order to prepare for tHESC electroporation | + | Midipreps of several different constructs in order to prepare for tHESC electroporation <br> <br> |
| − | 9/10/16 | + | 9/10/16 <br> |
| − | KB ARB | + | KB ARB <br> |
| − | Electroporation of tHESC | + | Electroporation of tHESC <br> <br> |
| − | 9/11/16 | + | 9/11/16 <br> |
| − | KB ARB | + | KB ARB <br> |
| − | Induction with E2 and P4 (note vehicle for E2 was 100% ethanol and for P4 was DMEM/F12) | + | Induction with E2 and P4 (note vehicle for E2 was 100% ethanol and for P4 was DMEM/F12) <br> <br> |
| − | 9/12/16 | + | 9/12/16 <br> |
| − | ARB SC KB | + | ARB SC KB <br> |
| − | Cytometry of tHESC induced samples | + | Cytometry of tHESC induced samples <br> <br> |
| − | 9/13/16 | + | 9/13/16 <br> |
| − | KB | + | KB <br> |
| − | Data analysis for cytometry experiment. | + | Data analysis for cytometry experiment. <br> <br> |
| − | 9/14/16 | + | 9/14/16 <br> |
| − | KB | + | KB <br> |
| − | Split MCF7 and seeded a well plate. | + | Split MCF7 and seeded a well plate. <br> <br> |
| − | 9/15/16 | + | 9/15/16 <br> |
| − | SC | + | SC <br> |
| − | Transfected MCF7 with promoter-repressor experiment | + | Transfected MCF7 with promoter-repressor experiment <br> <br> |
| − | 9/16/16 | + | 9/16/16 <br> |
| − | KB | + | KB <br> |
| − | Induced wells with estrogen. | + | Induced wells with estrogen. <br> <br> |
| − | 9/17/16 | + | 9/17/16 <br> |
| − | ARB | + | ARB <br> |
| − | Cytometry for the MCF7 repressor cascade experiment. | + | Cytometry for the MCF7 repressor cascade experiment. <br> |
| − | Split tHESC and seeded a well plate. | + | Split tHESC and seeded a well plate. <br> <br> |
| − | 9/18/16 | + | 9/18/16 <br> |
| − | ARB | + | ARB <br> |
| − | Electroporation of tHESC with pP4xeYFP, pHybrid constructs | + | Electroporation of tHESC with pP4xeYFP, pHybrid constructs <br> <br> |
| − | 9/19/16 | + | 9/19/16 <br> |
| − | ARB | + | ARB <br> |
| − | Induction of estrogen and progesterone on tHESC experiment | + | Induction of estrogen and progesterone on tHESC experiment <br> <br> |
| − | 9/20/16 | + | 9/20/16 <br> |
| − | ARB | + | ARB KB<br> |
| − | Flow cytometry of tHESC experiment | + | Flow cytometry of tHESC experiment <br> |
| − | + | Data analysis of results <br> <br> | |
| − | Data analysis of results | + | |
| − | 9/21/16 | + | 9/21/16 <br> |
| − | ARB | + | ARB <br> |
| − | Split MCF7 and seeded a well plate. | + | Split MCF7 and seeded a well plate. <br> <br> |
| − | 9/22/16 | + | 9/22/16 <br> |
| − | Electroporation of tHESC to test pERE3 with vehicle control. | + | ARB <br> |
| + | Electroporation of tHESC to test pERE3 with vehicle control. <br> <br> | ||
| − | 9/23/16 | + | 9/23/16 <br> |
| − | ARB | + | ARB <br> |
| − | Induction of pERE3 with estrogen. | + | Induction of pERE3 with estrogen. <br> <br> |
| − | 9/24/16 | + | 9/24/16 <br> |
| − | ARB KB | + | ARB KB <br> |
| − | Cytometry of tHESC vehicle test experiment for pERE3 | + | Cytometry of tHESC vehicle test experiment for pERE3 <br> <br> |
| − | 9/25/16 | + | 9/25/16 <br> |
| − | KB | + | KB <br> |
| − | Data analysis for tHESC experiment. | + | Data analysis for tHESC experiment. <br> |
| − | Split MCF7 and seeded a well plate. | + | Split MCF7 and seeded a well plate. <br> |
| − | Set up overnight colonies of hef1a-mKate for midiprepping. | + | Set up overnight colonies of hef1a-mKate for midiprepping. <br> <br> |
| − | 9/26/16 | + | 9/26/16 <br> |
| − | SC | + | SC <br> |
| − | Took out and spun down the midiprep colonies. | + | Took out and spun down the midiprep colonies. <br> |
| − | Transfected MCF7 with promoter-repressor experiment re-try | + | Transfected MCF7 with promoter-repressor experiment re-try <br> <br> |
| − | 9/27/16 | + | 9/27/16 <br> |
| − | KB | + | KB <br> |
| − | Induced cells with estrogen. | + | Induced cells with estrogen. <br> <br> |
| − | 9/28/16 | + | 9/28/16 <br> |
| − | KB ARB | + | KB ARB <br> |
| − | Flow cytometry for MCF7 experiment --failed due to low cell density and bad tfxn efficiency. | + | Flow cytometry for MCF7 experiment --failed due to low cell density and bad tfxn efficiency. <br> <br> |
| − | 9/29/16 | + | 9/29/16 <br> |
| − | ARB | + | ARB <br> |
| − | Split MCF7 cells with advice from Christi Cook on aseptic technique. | + | Split MCF7 cells with advice from Christi Cook on aseptic technique. <br> <br> |
| − | 9/30/16 | + | 9/30/16 <br> |
| − | KB | + | KB <br> |
| − | Data analysis for recombinase group -- bleed through correction. | + | Data analysis for recombinase group -- bleed through correction. <br> <br> |
| Line 443: | Line 460: | ||
| − | 10/6/16 | + | 10/6/16 <br> |
| − | KB ARB | + | KB ARB <br> |
| − | Split MCF7 and seeded a well plate. | + | Split MCF7 and seeded a well plate. <br> <br> |
| − | 10/7/16 | + | 10/7/16 <br> |
| − | KB | + | KB ARB<br> |
| − | Transfected MCF7 with final promoter experiment - PRE promoters, Hybrid | + | Transfected MCF7 with final promoter experiment - PRE promoters, Hybrid <br> |
| − | + | Electroporation of tHESC with final promoter experiment - PRE promoters fine induction, hybrid <br> <br> | |
| − | Electroporation of tHESC with final promoter experiment - PRE promoters fine induction, hybrid | + | |
| − | 10/8/16 | + | 10/8/16 <br> |
| − | KB | + | KB <br> |
| − | Induced wells with estrogen and progesterone. | + | Induced wells with estrogen and progesterone. <br> <br> |
| − | 10/9/16 | + | 10/9/16 <br> |
| − | KB ARB | + | KB ARB <br> |
| − | Imaged cells using the Zeiss | + | Imaged cells using the Zeiss <br> |
| − | Flow cytometry for tHESC and MCF7 experiment | + | Flow cytometry for tHESC and MCF7 experiment <br> |
| − | Data analysis of results | + | Data analysis of results <br> <br> |
</p> | </p> | ||
Revision as of 22:57, 18 October 2016
Notebook 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
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
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
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
