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
|