User:David K. Barclay/Notebook/Controlling Pancreas Cell Fate Using Transcription Factors/2014/04/15: Difference between revisions
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== | ==RT-PCR Mixes== | ||
* | '''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> | |||
|- | |||
| 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 H<sub>2</sub>O || (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'''<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 | |||
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Revision as of 12:53, 15 April 2014
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RT-PCR MixesReaction set-up: PCR master mixes for each Gene Target
*GAPD primer mix and the GAPD UPL probe are supplied in the Roche Universal ProbeLibrary Human GAPD Assay kit, #05190541001
Reaction set-up: master mixes for each Template
*For the no template control, use PCR H2O instead of cDNA. Resulting 1.5 mL tubes:
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