User:Karmella Haynes/Notebook/PcTF Genomics/2015/10/20: Difference between revisions

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==mm/dd/yy==
==10/20/15==
<!-- Precede finished items with a checkmark &#x2713; -->
<!-- Precede finished items with a checkmark &#x2713; -->
* Line item 1
* Luc/CFP swap - Colony growth


----
----
'''Line item 1'''<br>
'''Luc/CFP swap - Colony growth'''<br>
> Samples
* 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
* Today: plate cells at low concentration to try to get CFP-positive colonies


{| class="wikitable" border="0" cellspacing="3" <!-- Rxn. table -->
Images of transfected cells:
|-valign="top"
 
| <u>Reagent</u> || <u>Volume</u>
[[Image:someimage.jpg|400px]]
|-
 
| reagent 1 || # μL
Dilution procedure:
|-
# Wash with PBS and trypsinize in 0.5 mL trypsin medium
| reagent 2 || #
# Resuspend by adding 4.5 mL DMEM complete (+0.5 μg/mL puro) ≈ 200,000 cells/mL (assuming 1 million cells in 1 well, 6-well dish...there are probably fewer)
|-
# Add 0.5 mL resuspended cells to 10 mL medium in 10 cm plate ≈ 10,000 cells/mL
| reagent 3 || #
# Add 50 μL resuspended cells to 10 mL medium in 10 cm plate ≈ 1,000 cells/mL
|-
 
| reagent 4 || #
 
|-
Plates:
| dH<sub>2</sub>O || #
# Well 2
|-
| &nbsp; || # μL
|}


--> Reaction conditions




Revision as of 19:50, 20 October 2015

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10/20/15

  • Luc/CFP swap - Colony growth

Luc/CFP swap - Colony growth

  • 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 DBN007_pSB1A3 and (2) Cas9/gRNA34 plasmid
  • Today: plate cells at low concentration to try to get CFP-positive colonies

Images of transfected cells:

File:Someimage.jpg

Dilution procedure:

  1. Wash with PBS and trypsinize in 0.5 mL trypsin medium
  2. Resuspend by adding 4.5 mL DMEM complete (+0.5 μg/mL puro) ≈ 200,000 cells/mL (assuming 1 million cells in 1 well, 6-well dish...there are probably fewer)
  3. Add 0.5 mL resuspended cells to 10 mL medium in 10 cm plate ≈ 10,000 cells/mL
  4. Add 50 μL resuspended cells to 10 mL medium in 10 cm plate ≈ 1,000 cells/mL


Plates:

  1. Well 2




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