User:Karmella Haynes/Notebook/miR Trigger/2010/01/04

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01/04/10

  • RT-PCR: RFP reporter-transfected samples + controls

RT-PCR
> Round 1: Controls KAH108 (RFP), KAH108 +Dox (RFP, YFP), mock (untransfected)
--> Use dilutions of cDNA: 1. 1:10, 2. 1:100, 3. 1:1000, 4. 1:10000
--> 12 templates x 6 primer pairs YFP1, YFP2, RFP1, RFP2, MV2 (vector backbone), GAPDH
--> Make 13x master mix for each primer pair

Reagent Volume x13   Expected sizes = 100 bp
1-4. KAH108/YFP1
5-8. KAH108/YFP2
9-12. KAH108/RFP1
13-16. KAH108/RFP2
19-20. KAH108/MV2
21-24. KAH108/GAPDH
25-28. KAH108+Dox/YFP1
29-32. KAH108+Dox/YFP2
33-36. KAH108+Dox/RFP1
37-40.KAH108+Dox/RFP2
41-44. KAH108+Dox/MV2
45-48. KAH108+Dox/GAPDH
49-52. mock/YFP1
53-56. mock/YFP2
57-60. mock/RFP1
61-64. mock/RFP2
65-68. mock/MV2
69-72. mock/GAPDH
File:KAH 010410 gel1.tif
10 μL/lane, 1% agarose
cDNA 0.5 ---
10μM primer mix 2.0 26.0
2x GoTaq Green 10.0 130.0
dH2O 7.5 97.5
  20.0  
     
     
     
     
     
     

--> PCR: 95°C/ 3 min.; [95°C/ 30 sec, 57°C/ 30 sec, 72°C/ 30 sec] x 30; 72°C/ 3 min.; 4°C/ ∞


> Round 2: All samples
--> Use 1:1000 dilution of cDNA
--> 10 templates x 4 primer pairs: 1. YFP2, 2. RFP1, 3. GAPDH, 4. MV2 (vector backbone)
--> Make 11x master mix for each primer pair

Reagent Volume x11   Expected sizes = 100 bp
1. YFP2
2. RFP1
3. GAPDH
4. MV2
File:KAH 010410 gel2.tif
10 μL/lane, 1% agarose
cDNA 0.5 ---
10μM primer mix 2.0 22.0
2x GoTaq Green 10.0 110.0
dH2O 7.5 82.5
  20.0  
     
     
     
     
     
     

--> PCR: 95°C/ 3 min.; [95°C/ 30 sec, 57°C/ 30 sec, 72°C/ 30 sec] x 30; 72°C/ 3 min.; 4°C/ ∞