User:David K. Barclay/Notebook/Controlling Pancreas Cell Fate Using Transcription Factors/2014/04/15: Difference between revisions
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* Label one 1.5 mL tube per gene target | * Label one 1.5 mL tube per gene target | ||
* Make enough PCR master mix for your plate... | * Make enough PCR master mix for your plate... | ||
** ''' | ** '''PDX1''' is in Reactions 1, 8, and 15 = 3 | ||
** Replicates per reaction = 3 | ** Replicates per reaction = 3 | ||
** '''Master mix amount = 3 * 3 + 1 (to allow for pipetting error) = 10''' | ** '''Master mix amount = 3 * 3 + 1 (to allow for pipetting error) = 10''' | ||
** The same needs to be done for | ** The same needs to be done for MAFA, GLP1R, PCSK1, KCNQ2, IAPP and GAPD in separate tubes. | ||
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Resulting 1.5 mL tubes: | Resulting 1.5 mL tubes: | ||
* ''' | * '''PDX1''' - 85.0 μL | ||
* ''' | * '''MAFA''' - 85.0 μL | ||
* ''' | * '''GLP1R''' - 85.0 μL | ||
* ''' | * '''PCSK1''' - 85.0 μL | ||
* '''KCNQ2''' - 85.0 μL | |||
* '''IAPP''' - 85.0 μL | |||
* '''GAPD''' - 85.0 μL | * '''GAPD''' - 85.0 μL | ||
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* 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 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... | * Make enough Template master mix for your plate... | ||
** '''Treated cells cDNA''' is in Reactions 1, 2, 3, 4 | ** '''Treated cells cDNA''' is in Reactions 1, 2, 3, 4, 5, 6, 7 = 6 | ||
** Replicates per reaction = 3 | ** Replicates per reaction = 3 | ||
** '''Master mix amount = | ** '''Master mix amount = 7 * 3 + 1 (to allow for pipetting error) = 22''' | ||
** The same needs to be done for templates "untreated cells" and "no template" in separate tubes. | ** The same needs to be done for templates "untreated cells" and "no template" in separate tubes. | ||
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| <u>Reagent</u> || <u>(Single well)</u> || <u>cDNA Template (x16)</u> | | <u>Reagent</u> || <u>(Single well)</u> || <u>cDNA Template (x16)</u> | ||
|- | |- | ||
| 1:10 cDNA dilution || (2.0 μL) || | | 1:10 cDNA dilution || (2.0 μL) || 44.0* | ||
|- | |- | ||
| PCR H<sub>2</sub>O || (4.5 μL) || | | PCR H<sub>2</sub>O || (4.5 μL) || 99.0 | ||
|- | |- | ||
| Total vol. || ('''6.5 μL''') || ''' | | Total vol. || ('''6.5 μL''') || '''143.0''' | ||
|} | |} | ||
''*For the no template control, use PCR H<sub>2</sub>O instead of cDNA.'' | ''*For the no template control, use PCR H<sub>2</sub>O instead of cDNA.'' |
Revision as of 15:59, 18 April 2014
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