User:David K. Barclay/Notebook/Controlling Pancreas Cell Fate Using Transcription Factors/2014/04/04

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(Autocreate 2014/04/04 Entry for User:David_K._Barclay/Notebook/Controlling_Pancreas_Cell_Fate_Using_Transcription_Factors)
Current revision (13:24, 3 April 2014) (view source)
(Entry title)
 
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==Entry title==
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==RT-PCR Mixes==
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* Insert content here...
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'''Reaction set-up: PCR master mixes for each Gene Target'''<br>
 +
* Label one 1.5 mL tube per gene target
 +
* Make enough PCR master mix for your plate...
 +
** '''MPK14''' is in Reactions 1, 6, and 11 = 3
 +
** Replicates per reaction = 3
 +
** '''Master mix amount = 3 * 3 + 1 (to allow for pipetting error) = 10'''
 +
** The same needs to be done for CBX8, TNF, NPPA, and GAPD in separate tubes.
 +
 
 +
 
 +
{| class="wikitable"
 +
| <u>Reagent</u> || <u>(Single well)</u> || <u>Gene Target (x10)</u> || <u>GAPD (x10)</u>
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|-
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| 2x LC480 Probes Master || (7.5 μL) || 75.0 || 75.0
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|-
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| 20 μM Forward primer || (0.3 μL) || 3.0 || 3.0 GAPD primers*
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|-
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| 20 μM Reverse primer || (0.3 μL) || 3.0 || ---
 +
|-
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| 10 μM UPL probe || (0.3 μL) || 3.0 || 3.0 GAPD UPL probe*
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|-
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| PCR H<sub>2</sub>O || (0.1 μL) || 1.0 || '''4.0'''
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|-
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| Total vol. || ('''8.5 μL''') || '''85.0''' || '''85.0'''
 +
|}
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''*GAPD primer mix and the GAPD UPL probe are supplied in the Roche Universal ProbeLibrary Human GAPD Assay kit, #05190541001''
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 +
 
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Resulting 1.5 mL tubes:
 +
* '''MPK14''' - 85.0 μL
 +
* '''CBX8''' - 85.0 μL
 +
* '''TNF''' - 85.0  μL
 +
* '''NPPA''' - 85.0  μL
 +
* '''GAPD''' - 85.0  μL
 +
 
 +
----
 +
 
 +
'''Reaction set-up: master mixes for each Template'''<br>
 +
* Typically, you will have only 20 μL of stock cDNA on hand.
 +
* Make a 1:10 dilution of cDNA by adding 10 μL of the stock cDNA to 90 μL of PCR H<sub>2</sub>O.
 +
* Make enough Template master mix for your plate...
 +
** '''Treated cells cDNA''' is in Reactions 1, 2, 3, 4 and 5 = 5
 +
** Replicates per reaction = 3
 +
** '''Master mix amount = 5 * 3 + 1 (to allow for pipetting error) = 16'''
 +
** The same needs to be done for templates "untreated cells" and "no template" in separate tubes.
 +
 
 +
{| class="wikitable"
 +
| <u>Reagent</u> || <u>(Single well)</u> || <u>cDNA Template (x16)</u>
 +
|-
 +
| 1:10 cDNA dilution || (2.0 μL) || 32.0*
 +
|-
 +
| PCR H<sub>2</sub>O || (4.5 μL) || 72.0
 +
|-
 +
| Total vol. || ('''6.5 μL''') || '''104.0'''
 +
|}
 +
''*For the no template control, use PCR H<sub>2</sub>O instead of cDNA.''
 +
 
 +
Resulting 1.5 mL tubes:
 +
* '''T1''' - treated cells cDNA, 84.5 μL
 +
* '''T2''' - untreated cells cDNA, 84.5 μL
 +
* '''T3''' - no template control, 84.5  μL

Current revision

Cell Fate Switch by Synthetic Transcription Factors Main project page
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RT-PCR Mixes

Reaction set-up: PCR master mixes for each Gene Target

  • Label one 1.5 mL tube per gene target
  • Make enough PCR master mix for your plate...
    • MPK14 is in Reactions 1, 6, and 11 = 3
    • Replicates per reaction = 3
    • Master mix amount = 3 * 3 + 1 (to allow for pipetting error) = 10
    • The same needs to be done for CBX8, TNF, NPPA, and GAPD in separate tubes.


Reagent (Single well) Gene Target (x10) GAPD (x10)
2x LC480 Probes Master (7.5 μL) 75.0 75.0
20 μM Forward primer (0.3 μL) 3.0 3.0 GAPD primers*
20 μM Reverse primer (0.3 μL) 3.0 ---
10 μM UPL probe (0.3 μL) 3.0 3.0 GAPD UPL probe*
PCR H2O (0.1 μL) 1.0 4.0
Total vol. (8.5 μL) 85.0 85.0

*GAPD primer mix and the GAPD UPL probe are supplied in the Roche Universal ProbeLibrary Human GAPD Assay kit, #05190541001


Resulting 1.5 mL tubes:

  • MPK14 - 85.0 μL
  • CBX8 - 85.0 μL
  • TNF - 85.0 μL
  • NPPA - 85.0 μL
  • GAPD - 85.0 μL

Reaction set-up: master mixes for each Template

  • Typically, you will have only 20 μL of stock cDNA on hand.
  • Make a 1:10 dilution of cDNA by adding 10 μL of the stock cDNA to 90 μL of PCR H2O.
  • Make enough Template master mix for your plate...
    • Treated cells cDNA is in Reactions 1, 2, 3, 4 and 5 = 5
    • Replicates per reaction = 3
    • Master mix amount = 5 * 3 + 1 (to allow for pipetting error) = 16
    • The same needs to be done for templates "untreated cells" and "no template" in separate tubes.
Reagent (Single well) cDNA Template (x16)
1:10 cDNA dilution (2.0 μL) 32.0*
PCR H2O (4.5 μL) 72.0
Total vol. (6.5 μL) 104.0

*For the no template control, use PCR H2O instead of cDNA.

Resulting 1.5 mL tubes:

  • T1 - treated cells cDNA, 84.5 μL
  • T2 - untreated cells cDNA, 84.5 μL
  • T3 - no template control, 84.5 μL



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