User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/06/06: Difference between revisions

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">PcTF Subcloning in E. coli</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">PcTF Subcloning in E. coli</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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==06/06/2013==
==06/06/2013==
<!-- Precede each finished task with a checkmark &#x2713; -->
* &#x2713; Minipreps: FlpE (4)
----
'''Minipreps'''<br>
> Check with E, P, and E/P digests
{| {{table}} cellspacing="7"  width=700px
|-valign="top"
| <u>Reagent</u> || <u>1,4,7,10</u> || <u>2,5,8,11</u> || <u>3,6,9,12</u>
| rowspan="7" | <u>Expected:</u><br>1. FlpE, E = 7160, 546<br>2. FlpE, P = 7706<br>3.FlpE, E/P=5649, 1511, 546
| rowspan="7" | [[Image:Flp-E.jpg|300px|E/P digests 05/06/10]]<br>15 μL/lane; 1% agarose
|-
| DNA(plasmid) || 2.0 || 2.0 || 2.0
|-
| 10X buffer || 1.5 || 1.5 || 1.5
|-
| EcoRI || 1.0 || --- || 1.0
|-
| PstI || --- || 1.0 || 1.0
|-
| dH<sub>2</sub>O || 10.5 || 10.5 || 9.5
|-
| &nbsp;<br>&nbsp;<br>&nbsp;<br>&nbsp;||
|}
15 μL --> 37°C/ ~15 min.
--> Conclusion: Success! Combine 4 preps (300μL total) for DNA clean-up (tomorrow)<br>
--> Streak clone (saved toothpick) onto 100 μg/mL Amp plate.<br>




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----
----
'''1 day before transfection:'''<br>
# Seed cultures in 6-well plates at ~2.5x10<suP>5</sup> so that cells will be ~5.0x10<sup>5</sup> at the time of transfection.
'''Transfections'''<br>
'''Transfections'''<br>
> U2OS Flp-in T-REx lines (puro/zeo/blast) + FRT-marked mammalian transfection vector (hygro)<br>
> U2OS Flp-in T-REx lines (puro/zeo/blast) + FRT-marked mammalian transfection vector (hygro)<br>
--> Note: co-transfect with FlpE (1:3); hygro resistance replaces zeo resistance after "Flp" in<br>
--> Note: co-transfect with FlpE (1:3); hygro resistance replaces zeo resistance after "Flp" in<br>
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BD002 (3:1) = 339 ng/μl
BD002 (3:1) = 339 ng/μl
BD002 (4:1) = 317 ng/μl
BD002 (4:1) = 317 ng/μl
PlpE = 192 ng/μl
KAH187 = 441.5 ng/μl
FlpE = 192 ng/μl


{| {{table}} cellspacing="7"  width=700px  
{| {{table}} cellspacing="7"  width=700px  
|-
|-
| <u>Plate-Well</u> || <u>Plasmid</u> || <u>DNA</u> || <u>Volume</u>|| dH2O || <u>Lipo</u>  || <u>Opti-MEM (total)</u>
| Plate-Well || Plasmid || DNA || Volume|| dH2O|| Lipo || PLUS reagent || <u>Opti-MEM (total)</u>
|-
| 1-1            || BD002 (3:1) + FlpE  || 1.5 + 0.5 μg || 4.42 + 2.60 μL || 12.98μL|| 4 μL      || 500 μL
|-
|-
| 1-2           || BD002 (3:1) + FlpE || "          || 4.42 + 2.60 μL   || 12.98μL|| "            || "
| 1-1           || BD002 (3:1) + FlpE || 1.5 + 0.5 μg || 4.42 + 2.60 μL || 12.98μL|| 2.5μL|| 7.5 μL      || 570 μL
|-
|-
| 1-3           || BD002 (4:1) + FlpE || "          || 4.73 + 2.60 μL || 12.67μL || "             || "
| 1-2           || BD002 (4:1) + FlpE || "          || 4.73 + 2.60 μL   || 12.67μL|| " ||    "      || "
|-
|-
| 1-4            || BD002 (4:1)+ FlpE || "          || 4.73 + 2.60 μL  || 12.67μL|| "             || "
| 1-3 (-) Ctrl    || BD002 (3:1) + --- || 2 μg      || 4.42 + --- μL  || 15.58μL || "    ||   "     || "
|-
|-
|1-5            || BD002 (3:1) + --- ||           ||   4.42 + ---  || 15.58μL||       ||
| 1-4 (-)Ctrl      || --- + --- || 0 μg        || --- + --- μL || 20.0μL|| "    ||   "    || "
|-
|-
| 1-6             || BD002 (4:1) + ---  ||             || 4.73 + ---  || 15.58μL ||     ||
|1-5             || KAH187    ||   20 μg        ||   4.53  || 15.46μL||    "  || "||"
|}
|}


# Prepare DNA-Lipofectamine complexes (600 μl per sample) as follows:
## Label sterile microfuge (1.5 ml) tubes.
## Add '''570 μL Opti-MEM''' to each 20 μL DNA sample.
## Add '''2.5 μL PLUS reagent''' to each DNA+Opti-MEM sample. Incubate for 5 minutes at room temperature.
## Add '''7.5 μL Lipofectamine LTX''' to each DNA+Opti-MEM+PLUS reagent sample. Incubate for 30 minutes at room temperature.
# Add the total 600 μL of DNA+Opti-MEM+PLUS reagent+Lipofectamine mixture '''drop-wise''' to each appropriate well of cells.
# Incubate cells at 37°C in a CO<sub>2</sub> incubator
# (Optional) To reduce toxicity, after 5-6 hours wash cells once with warm 1xPBS to remove excess DNA-Lipo complexes. Add fresh antibiotic-free growth medium.
Transgene expression should be detectable after 18 hours.


> Add 16 μL (4x) Lipo to 1000 μL (4x) Opti-MEM --> R.T/ 5 min. <br>
> Add DNA to 250 μL Opti-MEM <br>
> Add 250 Lipo mix into each 250 DNA mix --> R.T./ 20 min. <br>
> Add 500 μL complexes to each 3.5 mL well (4 ml med. total each); Grow cells at 37&deg;C<br>
> Refresh medium after 5 hours (ab-free)<br>
> Grow for one/two days, then plate dilution cultures in selection medium (+hygro)




----
----
'''Polycomb-ATF negative control lines: colony plates'''<br>
> For each line make 2 plates: ~1x10<sup>5</sup> and 5x10<sup>4</sup> cells<br>
> 25 ml 1 μg/mL puro, 15 μg/mL blast, 200 μg/mL zeo<br>
> Also make 6-well back-up plate for all transfections (~3x10<sup>5</sup> cells in selection medium)
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
|}


__NOTOC__
__NOTOC__

Latest revision as of 22:45, 26 September 2017

PcTF Subcloning in E. coli Main project page
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06/06/2013

  • ✓ Minipreps: FlpE (4)

Minipreps
> Check with E, P, and E/P digests

Reagent 1,4,7,10 2,5,8,11 3,6,9,12 Expected:
1. FlpE, E = 7160, 546
2. FlpE, P = 7706
3.FlpE, E/P=5649, 1511, 546 		E/P digests 05/06/10
15 μL/lane; 1% agarose
DNA(plasmid) 2.0 2.0 2.0
10X buffer 1.5 1.5 1.5
EcoRI 1.0 --- 1.0
PstI --- 1.0 1.0
dH2O 10.5 10.5 9.5
 
 
 
 

15 μL --> 37°C/ ~15 min.


--> Conclusion: Success! Combine 4 preps (300μL total) for DNA clean-up (tomorrow)
--> Streak clone (saved toothpick) onto 100 μg/mL Amp plate.


  • ✓ Transfections: H3K27me3 reporters into cell lines BD002 (KAH201+KAH182+V0200)
  • ✓ Polycomb-ATF negative control lines: colony plates


1 day before transfection:

  1. Seed cultures in 6-well plates at ~2.5x105 so that cells will be ~5.0x105 at the time of transfection.

Transfections

> U2OS Flp-in T-REx lines (puro/zeo/blast) + FRT-marked mammalian transfection vector (hygro)
--> Note: co-transfect with FlpE (1:3); hygro resistance replaces zeo resistance after "Flp" in
> Use Lipofectamine, 6-well format
> Plates:

  1. BD002 (3:1) Flp-in T-REx
  2. BD002 (4:1) Flp-in T-REx

BD002 (3:1) = 339 ng/μl BD002 (4:1) = 317 ng/μl KAH187 = 441.5 ng/μl FlpE = 192 ng/μl

Plate-Well Plasmid DNA Volume dH2O Lipo PLUS reagent Opti-MEM (total)
1-1 BD002 (3:1) + FlpE 1.5 + 0.5 μg 4.42 + 2.60 μL 12.98μL 2.5μL 7.5 μL 570 μL
1-2 BD002 (4:1) + FlpE " 4.73 + 2.60 μL 12.67μL " " "
1-3 (-) Ctrl BD002 (3:1) + --- 2 μg 4.42 + --- μL 15.58μL " " "
1-4 (-)Ctrl --- + --- 0 μg --- + --- μL 20.0μL " " "
1-5 KAH187 20 μg 4.53 15.46μL " " "
  1. Prepare DNA-Lipofectamine complexes (600 μl per sample) as follows:
    1. Label sterile microfuge (1.5 ml) tubes.
    2. Add 570 μL Opti-MEM to each 20 μL DNA sample.
    3. Add 2.5 μL PLUS reagent to each DNA+Opti-MEM sample. Incubate for 5 minutes at room temperature.
    4. Add 7.5 μL Lipofectamine LTX to each DNA+Opti-MEM+PLUS reagent sample. Incubate for 30 minutes at room temperature.
  2. Add the total 600 μL of DNA+Opti-MEM+PLUS reagent+Lipofectamine mixture drop-wise to each appropriate well of cells.
  3. Incubate cells at 37°C in a CO2 incubator
  4. (Optional) To reduce toxicity, after 5-6 hours wash cells once with warm 1xPBS to remove excess DNA-Lipo complexes. Add fresh antibiotic-free growth medium.

Transgene expression should be detectable after 18 hours.



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