Haynes Lab:Notebook/CRISPR Editing/2015/06/30: Difference between revisions

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==03//2015==
==06/30/2015==
transfected Luc14s gal4-eeds with dox with 2 dilutions of g034 in triplicate.<br><br>
'''Step 1: Diluting the DNA'''
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''ug of DNA for 3.2 wells'''
| align="center" style="background:#f0f0f0;"|'''ul plasmid'''
| align="center" style="background:#f0f0f0;"|'''water (32ul total)'''
|-
| g034 0.1|0.32||||
|-
| g034 0.01||0.032||||
|-
| g034 0.1||0.32||||
|-
| g034 0.01||0.032||||
|}
<br>
'''Step 2: make DNA + optimem mixes'''
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''1 well'''
| align="center" style="background:#f0f0f0;"|'''3.2 x'''
|-
| g034 0.25||39||add 124.8ul optimem
|-
| g034 0.5||39||add 124.8ul optimem
|-
| g034 0.75||39||add 124.8ul optimem
|-
| g034 1.0||39||add 124.8ul optimem
|}
<br>
'''Step 3: add plus reagent to DNA+ opti'''
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''1 well'''
| align="center" style="background:#f0f0f0;"|'''3.2 x'''
|-
| g034 0.25||1||add 3.2ul plus reagent
|-
| g034 0.5||1||add 3.2ul plus reagent
|-
| g034 0.75||1||add 3.2ul plus reagent
|-
| g034 1.0||1||add 3.2ul plus reagent
|}
<br>
'''Step 4: Make optimem + lipo mastermix'''
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''1 well'''
| align="center" style="background:#f0f0f0;"|'''for 12 wells (x 13.2)'''
|-
| optimem||47||620.4
|-
| lipo||3||39.6
|}
<br>
'''Step 5: mix dna mix and lipo mix'''
{| {{table}}
| g034 0.25||add 160 lipo mix
|-
| g034 0.5||add 160 lipo mix
|-
| g034 0.75||add 160 lipo mix
|-
| g034 1.0||add 160 lipo mix
|}
'''Step 6: add 100ul to each well'''


<br><br>
Collected transfected gal4-eed cells with dox or puro for flow and gDNA extraction:
<br>
:aspirate media
:add 200ul PBS, rock plate, aspirate PBS
:add 100ul trypsin, let sit for a few minutes, tap plate to unstick cells
:add 400ul media, pipette to collect cells, move to 1.5mL microcentrifuge tube
:remove tubes from hood
:spin in benchtop centrifuge at 0.2x1000RCF for 3 minutes
:remove media with P1000
:resuspend in 400ul cold PBS
:move 200ul to 2mL tube for flow, use remaining 200ul for gDNA extraction
<br>




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06/30/2015

transfected Luc14s gal4-eeds with dox with 2 dilutions of g034 in triplicate.

Step 1: Diluting the DNA

' ug of DNA for 3.2 wells ul plasmid water (32ul total)
0.32
g034 0.01 0.032
g034 0.1 0.32
g034 0.01 0.032


Step 2: make DNA + optimem mixes

' 1 well 3.2 x
g034 0.25 39 add 124.8ul optimem
g034 0.5 39 add 124.8ul optimem
g034 0.75 39 add 124.8ul optimem
g034 1.0 39 add 124.8ul optimem


Step 3: add plus reagent to DNA+ opti

' 1 well 3.2 x
g034 0.25 1 add 3.2ul plus reagent
g034 0.5 1 add 3.2ul plus reagent
g034 0.75 1 add 3.2ul plus reagent
g034 1.0 1 add 3.2ul plus reagent


Step 4: Make optimem + lipo mastermix

' 1 well for 12 wells (x 13.2)
optimem 47 620.4
lipo 3 39.6


Step 5: mix dna mix and lipo mix

g034 0.25 add 160 lipo mix
g034 0.5 add 160 lipo mix
g034 0.75 add 160 lipo mix
g034 1.0 add 160 lipo mix

Step 6: add 100ul to each well



Collected transfected gal4-eed cells with dox or puro for flow and gDNA extraction:

aspirate media
add 200ul PBS, rock plate, aspirate PBS
add 100ul trypsin, let sit for a few minutes, tap plate to unstick cells
add 400ul media, pipette to collect cells, move to 1.5mL microcentrifuge tube
remove tubes from hood
spin in benchtop centrifuge at 0.2x1000RCF for 3 minutes
remove media with P1000
resuspend in 400ul cold PBS
move 200ul to 2mL tube for flow, use remaining 200ul for gDNA extraction




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