User:Karmella Haynes/Notebook/PcTF Genomics/2015/06/27: Difference between revisions

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(Autocreate 2015/06/27 Entry for User:Karmella_Haynes/Notebook/PcTF_Genomics)
 
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==mm/dd/yy==
==mm/dd/yy==
<!-- Precede finished items with a checkmark &#x2713; -->
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* Line item 1
* Cas-tone - cell transfections (Gal4EED/luc)
 


----
----
'''Line item 1'''<br>
'''Cas-tone - transfections'''<br>
> Samples
* Final H3#_MV11 builds verified on 6/24/15
* Co-transfect H3#_MV11 with pSP-guide RNA plasmid (built by Ben Nyer)
* Use HEK293 Gal4EED/luc cells, no dox (from Rene)
 
# H340_MV11 + g034_pSPgRNA
# H340_MV11 (no gRNA)
# H360_MV11 + g034_pSPgRNA
# H360_MV11 (no gRNA)
# H380_MV11 + g034_pSPgRNA
# H380_MV11 (no gRNA)


{| class="wikitable" border="0" cellspacing="3" <!-- Rxn. table -->
{| {{table}} border="0" cellspacing="3" <!-- Trfxn. table -->
|-valign="top"
|-valign="top"
| <u>Reagent</u> || <u>Volume</u>  
| <u>Well</u> || <u>H3#_MV11 (1.0 μg)</u> || <u>g034_pSPgRNA (1.0 μg)</u> || <u>dH<sub>2</sub>O</u> || <u>OptiMEM</u> || <u>PLUS rgt.</u> || <u>Lipo LTX</u>
|-
|-
| reagent 1 || # μL
| 1 || 5.0 || 1.6 || 13.4 || 570.0 || 2.5 || 7.5
|-
|-
| reagent 2 || #
| 2 || 5.0 || --- || 15.0 || 570.0 || 2.5 || 7.5
|-
|-
| reagent 3 || #
| 3 || 5.0 || 1.6 || 13.4 || 570.0 || 2.5 || 7.5
|-
|-
| reagent 4 || #
| 4 || 5.0 || --- || 15.0 || 570.0 || 2.5 || 7.5
|-
|-
| dH<sub>2</sub>O || #
| 5 || 5.6 || 1.6 || 12.8 || 570.0 || 2.5 || 7.5
|-
|-
| &nbsp; || # μL
| 6 || 5.6 || --- || 14.4 || 570.0 || 2.5 || 7.5
|}
|}


--> Reaction conditions
* Pre-warm ps-free DMEM
* Make 20 μL plasmid/dH<sub>2</sub>O mixes in sterile 1.5 ml tubes
* Add OptiMEM
* Add PLUS --> 5 min./ RT
* Add Lipo LTX --> 30 min./ RT
* Cells - refresh growth medium w/ ps-free DMEM
* Add 600 μL complexes, drop-wise
* Incubate overnight




Revision as of 18:25, 30 June 2015

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mm/dd/yy

  • Cas-tone - cell transfections (Gal4EED/luc)


Cas-tone - transfections

  • Final H3#_MV11 builds verified on 6/24/15
  • Co-transfect H3#_MV11 with pSP-guide RNA plasmid (built by Ben Nyer)
  • Use HEK293 Gal4EED/luc cells, no dox (from Rene)
  1. H340_MV11 + g034_pSPgRNA
  2. H340_MV11 (no gRNA)
  3. H360_MV11 + g034_pSPgRNA
  4. H360_MV11 (no gRNA)
  5. H380_MV11 + g034_pSPgRNA
  6. H380_MV11 (no gRNA)
Well H3#_MV11 (1.0 μg) g034_pSPgRNA (1.0 μg) dH2O OptiMEM PLUS rgt. Lipo LTX
1 5.0 1.6 13.4 570.0 2.5 7.5
2 5.0 --- 15.0 570.0 2.5 7.5
3 5.0 1.6 13.4 570.0 2.5 7.5
4 5.0 --- 15.0 570.0 2.5 7.5
5 5.6 1.6 12.8 570.0 2.5 7.5
6 5.6 --- 14.4 570.0 2.5 7.5
  • Pre-warm ps-free DMEM
  • Make 20 μL plasmid/dH2O mixes in sterile 1.5 ml tubes
  • Add OptiMEM
  • Add PLUS --> 5 min./ RT
  • Add Lipo LTX --> 30 min./ RT
  • Cells - refresh growth medium w/ ps-free DMEM
  • Add 600 μL complexes, drop-wise
  • Incubate overnight



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