Haynes Lab:Notebook/CRISPR Editing/2015/03/06: Difference between revisions

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}}
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<br>
plasmid ng/µL ul for 05.ng
g044 403.212 1.240042459
g031 288.187 1.734984576
<br><br>
plated cells look great, all wells are about 90% confluent. Will proceed with all 48 transfections
<br><br>
'''KAH60 transfections'''


{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Step 1'''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
|-
| ||||||||
|-
| ||Making 1ug/10ul plasmid stocks||||||
|-
| ||||ng/ul||ul plasmid||ul H2O
|-
| ||KAH60||278.411||51.00||91.00
|-
| ||pMax GFP 6.2||551.779||14.86102226||75.13897774
|-
| ||g032||452.739||13.47||48.53
|-
| ||g031||288.187||21.17||40.83
|-
| ||g025||235.95||25.85||36.15
|-
| ||g044||403.212||15.13||46.87
|-
| ||||||||
|-
| Step 2||||||||
|-
| ||make DNA + optimem mixes||||||
|-
| ||||||||
|-
| ||||||||
|-
| ||KAH60||add 554 ul optimem||||
|-
| ||pMax GFP 6.2||add 320 ul optimem||||
|-
| ||g032||add 242 ul optimem||||
|-
| ||g031||add 242 ul optimem||||
|-
| ||g025||add 242 ul optimem||||
|-
| ||g044||add 242 ul optimem||||
|-
| ||||||||
|-
| Step 3||||||||
|-
| ||add plus reagent to DNA+ opti||||||
|-
| ||||||||
|-
| ||KAH60||add 14.2ul plus||||
|-
| ||pMax GFP 6.2||add 8.2ul plus||||
|-
| ||g032||add 6.2ul plus||||
|-
| ||g031||add 6.2ul plus||||
|-
| ||g025||add 6.2ul plus||||
|-
| ||g044||add 6.2ul plus||||
|-
| ||||||||
|-
| ||||||||
|-
| step 4||||||||
|-
| ||Make optimem + lipo mastermix||||||
|-
| ||||||||
|-
| ||Optimem||2265.4||||
|-
| ||lipo||144.6||||
|-
| ||||||||
|-
| ||||||||
|-
| Step 5||||||||
|-
| ||||||||
|-
| ||mix dna mix and lipo mix||||||
|-
| ||||||||
|-
| ||KAH60||add 710 lipo mix||||
|-
| ||pMax GFP 6.2||add 410 lipo mix||||
|-
| ||g032||add 310 lipo mix||||
|-
| ||g031||add 310 lipo mix||||
|-
| ||g025||add 310 lipo mix||||
|-
| ||g044||add 310 lipo mix||||
|-
| ||||||||
|-
| Step 6||||||||
|-
| ||||||||
|-
| ||add 100ul dropwise to each well||||||
|}
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__NOTOC__
__NOTOC__

Latest revision as of 00:48, 27 September 2017

Project name Main project page
Previous entry      Next entry

03/06/2015

miniprep of pMax GFP 6.2 from Lonza and KAH60

plasmid colony 260 280 260/280 ng/µL ul for 05.ng
pMax GFP 6.2 1 0.552 0.299 1.848 551.779 0.906159894
pMax GFP 6.2 2 0.348 0.187 1.861 347.553 1.438629504
KAH60 1 0.102 0.056 1.817 101.806 4.911301888
KAH60 2 0.278 0.148 1.878 278.411 1.795906053


plasmid ng/µL ul for 05.ng g044 403.212 1.240042459 g031 288.187 1.734984576

plated cells look great, all wells are about 90% confluent. Will proceed with all 48 transfections

KAH60 transfections


Step 1 ' ' ' '
Making 1ug/10ul plasmid stocks
ng/ul ul plasmid ul H2O
KAH60 278.411 51.00 91.00
pMax GFP 6.2 551.779 14.86102226 75.13897774
g032 452.739 13.47 48.53
g031 288.187 21.17 40.83
g025 235.95 25.85 36.15
g044 403.212 15.13 46.87
Step 2
make DNA + optimem mixes
KAH60 add 554 ul optimem
pMax GFP 6.2 add 320 ul optimem
g032 add 242 ul optimem
g031 add 242 ul optimem
g025 add 242 ul optimem
g044 add 242 ul optimem
Step 3
add plus reagent to DNA+ opti
KAH60 add 14.2ul plus
pMax GFP 6.2 add 8.2ul plus
g032 add 6.2ul plus
g031 add 6.2ul plus
g025 add 6.2ul plus
g044 add 6.2ul plus
step 4
Make optimem + lipo mastermix
Optimem 2265.4
lipo 144.6
Step 5
mix dna mix and lipo mix
KAH60 add 710 lipo mix
pMax GFP 6.2 add 410 lipo mix
g032 add 310 lipo mix
g031 add 310 lipo mix
g025 add 310 lipo mix
g044 add 310 lipo mix
Step 6
add 100ul dropwise to each well


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