User:Karmella Haynes/Notebook/PcTF Genomics/2015/10/20: Difference between revisions
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'''Luc/CFP swap - Colony growth'''<br> | '''Luc/CFP swap - Colony growth'''<br> | ||
* 10/19/15 - Ben Nyer - Gal4EED/luc HEK293 cells were co-transfected with (1) HPK-CFP gene flanked with homology arms that match luciferase site [https://benchling.com/s/IxMj4FhL/edit DBN007_pSB1A3] and (2) Cas9/gRNA34 plasmid | * 10/19/15 - Ben Nyer - Gal4EED/luc HEK293 cells were co-transfected with lipofectamine LTX and (1) HPK-CFP gene flanked with homology arms that match luciferase site [https://benchling.com/s/IxMj4FhL/edit DBN007_pSB1A3] and (2) Cas9/gRNA34 plasmid | ||
* Today: plate cells at low concentration to try to get CFP-positive colonies | * Today: plate cells at low concentration to try to get CFP-positive colonies | ||
Images of transfected cells in 6-well dish: | |||
[[Image:KAH102015 cells1.jpg|500px]] | [[Image:KAH102015 cells1.jpg|500px]] | ||
Expansion procedure (to maintain back-up stocks): | |||
# Wash with PBS and trypsinize in 0.5 mL trypsin medium | |||
# Resuspend with 3.5 mL DMEM complete (+0.5 μg/mL puro); add to 6 mL DMEM complete (+0.5 μg/mL puro) in 10 cm dish | |||
Dilution procedure: | Dilution procedure: | ||
# Wash with PBS and trypsinize in 0.5 mL trypsin medium | # Wash with PBS and trypsinize in 0.5 mL trypsin medium | ||
# Resuspend by adding 4.5 mL DMEM complete (+0.5 μg/mL puro) ≈ 200,000 cells/mL | # Assume 1 million cells in 1 well...there are probably fewer) ≈ 1,000,000 cells | ||
# Resuspend by adding 4.5 mL DMEM complete (+0.5 μg/mL puro) ≈ 200,000 cells/mL | |||
# Add 0.5 mL resuspended cells to 10 mL medium in 10 cm plate ≈ 10,000 cells/mL | # Add 0.5 mL resuspended cells to 10 mL medium in 10 cm plate ≈ 10,000 cells/mL | ||
# Add 50 μL resuspended cells to 10 mL medium in 10 cm plate ≈ 1,000 cells/mL | # Add 50 μL resuspended cells to 10 mL medium in 10 cm plate ≈ 1,000 cells/mL | ||
Plates: | |||
# Well 2 | Plates set up today: | ||
# Luc/CFP Well 1 (expansion) | |||
# Luc/CFP Well 2 1:100 (10,000 cells/mL) | |||
# Luc/CFP Well 2 1:1000 (1,000 cells/mL) | |||
# Luc/CFP Well 3 1:100 (10,000 cells/mL) | |||
# Luc/CFP Well 3 1:1000 (1,000 cells/mL) | |||
# Luc/CFP Well 4 (expansion) | |||
# Luc/CFP Well 5 (expansion) | |||
Revision as of 20:00, 20 October 2015
 Pc-TF Genomics
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10/20/15
Luc/CFP swap - Colony growth
Images of transfected cells in 6-well dish:
Plates set up today:
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