Embedded Files
Cell Culture Project
Cell Culture Project
Google Slide Presentation Outlining Basics of CRISPR Project and Cell Culture in our Lab Currently
The long-range goal of the Cell Culture project is to edit the cystic fibrosis (CF) gene in Calu-3 and human bronchial epithelial cells using CRISPR, and also demonstrate the blockade of the binding between SARS-CoV-2 Spike Protein and human ACE2 in Calu-3 and human bronchial epithelial cells with the use of a designed decoy receptor.
What you can do to help the team
Help us with cell culture. This includes processes such as cell passage and transfection. (Precise/Technical Learning Pattern)
Help us with literature review. (Precise Learning Pattern)
Help us by designing the CRISPR 3.0 plasmid (Precise/Technical/Confluent Learning Pattern)
Help us by designing the plasmids for RBD-IgG, and ACE2-mRuby fusion proteins.
Cell Culture Project Materials and DNA: List and Locations
Project progress so far
Project progress so far
CRISPR Project Progress List
CRISPR Project Progress List
Tasks Completed
Acquiring Cells
Acquired LM45 Cell Line from Dr. Baer (Fall 2019)
Acquired LM45-CD47 Cell Line from Dr. Baer (Spring 2020)
This line of cells permanently expressed eGFP, and can therefore be used at any point to practice observing fluorescence with the microscope in Room 515
Passaging Cells
Passage LM45 Cells Once every ~7 days (Fall 2019- Present)
Passage LM45-CD47 Cell Line once every ~7 Days (January 2020 - Present)
Transfecting Cells
Successfully Transfected eGFP into LM45 Cells, and likely transfected Cas9 (Dr. Liu) into LM45 Cells (Fall 2019, and Spring 2020)
Freezing Cells
3 Tubes of LM45 Cells Freezing at -80 (2/2020)
1 Tube of LM45-CD47 Freezing at -80 (2/2020)
Unthawing Cells
1 Tube of previously frozen LM45 Cells has was successfully unthawed and used to plate 2 60 mm dishes (original frozen tube was made from 1 60mm dish of 100% confluent LM45 cells) (2/2020)
Inventory (Link to Inventory List)
DNA
Cas9 received in plasmid form DNAsu. Glycerol stock and DNA freezing at -80 C (3/3/2020)
Cas9 (from Dr. Liu) used for CFTR editing in Freezer A
eGFP DNA in Freezer A
Solutions/Media/Reagents
Lipofectamine Transfection Kit located in Fridge B
RPMI 1640 Located in Fridge B
RPMI 1640 + 10% FBS located in Fridge A
Trypsin ocated in Fridge A
Equipment
Mr Frosty in cabinet above oocyte injection station
Current Number Of Freezes = 3 (replace alcohol after 5) (as of 3/3/2020)
24 well plates located in Large Box by Centrifuge near oocyte injection station
Hemocytomers Located in FedEx Package on 24 Well Plate Box
Link to other CRISP Project Inventory Here
Currently In Progress
CRISPR Project is currently suspended due to COVID Pandemic
Researching Anti-Cancer Drug of choice (Current = Vemurafenib) to assess effectiveness on cultured cells
Designing Guide RNA to use in conjunction with Cas9 enzyme ordered from DNAsu.
Current chosen Target is B-Raf
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