Haynes Lab:Notebook/Synthetic Biology and Bioinformatics for Predictable Control of Therapeutic Gene2/2012/11/29: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
Caroline Hom (talk | contribs) (Autocreate 2012/11/29 Entry for Haynes_Lab:Notebook/Synthetic_Biology_and_Bioinformatics_for_Predictable_Control_of_Therapeutic_Gene2) |
Caroline Hom (talk | contribs) |
||
Line 6: | Line 6: | ||
| colspan="2"| | | colspan="2"| | ||
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | ||
== | ==Transfection of K562== | ||
* | * Cell Density if at 1 mil/mg | ||
* Target Concentration of 500,000 cells/mL per well (6 well plate) | |||
* We need about 11uL (2000ng DNA/DNA sample in wells 1, 3, and 5) | |||
'''Part 1''' | |||
#Pellet cells to get rid of PS, aspirate (re-suspend 10mL into 15mL conical vial in PS free, spin for 3 min) | |||
#Dilution (1/2, 1/4, 1/8) - make sure to pipet up and down | |||
#Incubate while setting up transfection rxn | |||
'''Part 2''' | |||
#Transfer 4mL of OPTI-MEM to a 15mL conical vial (write on opti-mem when it was opened and initials; label opti-mem tube; make sure opti-mem, lipofectamine, and PLUS Reagent are at room temp) | |||
#Add 570uL OPTI-MEM to sames and flick tube/invert | |||
#Add 2.5uL PLUS Reagent to samples, incubate 5 min @ room temp | |||
#Add 7.5uL of lipofectamine to the amples, incubate @ room temp for 30 min | |||
#Add solution drop by drop ti each well | |||
#Incubate (label wells w/ #'s and +/- DNA) | |||
'''Wells''' | |||
# 5 x 10^5 cells DNA (11uL DNA, 9uL H2O) | |||
# 5 x 10^5 cells NEG (20uL H2O) | |||
# 2.5 x 10^5 cells DNA (11uL DNA, 9uL H2O) | |||
# 2.5 x 10^5 cells NEG (20uL H2O) | |||
# 1.25 x 10^5 cells DNA (11uL DNA, 9uL H2O) | |||
# 1.25 x 10^5 cells NEG (20uL H2O) | |||
Revision as of 01:05, 20 December 2012
Project name | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> |
Transfection of K562
Part 1
Part 2
Wells
|