Spectrum of Innovation CHROMOUS BIOTECH PVT. LD. PCR 12 RT-PCR Kit (MMULYV, Single tube) The Kit contains sufficient reagents to carry «nut 20 independent RT-PCR reactions. The Kit contains MMuLV Reverse Transerip ase to synthesize firststrand cDNA and high “fidelity polymerase for PCR (ChromTaq pols merase). * Control RNA and control primers (gene spec fic) are provided to carry out 3 control reactions Kit components: 1. Reaction Mix 575 pl 2. Enzyme Mix 50 ll 3. Control Reverse Primer (100ng/tI) 12 4. Control Forward Primer (100ng/ ll): 6 ul 5. Control RNA: 5 ug |provided as dry pellet) (Add 50 jul DEPC water and incubate at 65°C for 15 min to suspend the RNA) 6. DEPC water: 1 ml Storage: All components except RNA have to be stored at -20°C. Store RNA at -70°C. Thaw all the components on ice before starting the experiment. Protocol: For Control reactions: 1. Mix 5 pl of control RNA Template (1 jig) and 4 tl of Reverse Primer, 3 4 of forward Primer Add 11 j1l of DEPC water to the reaction mix, 2. Heat inactivate at 65°C for 10 min and Chill on ice. 3. Set-up Control RT PCR reactions by adding the components in the following order * Add 25 ull Reaction mix provided * Add 23 ull RNA- primer mix and finally add 2 \1l enzyme mix 4, Set-up RT PCR reaction RT PCR Cycle Conditions for Control Reaction only: [42°C 98°C - | 30min | 15min | — > Load 10 Hl of PCR product, Expected Control reaction product: 400 bp No.842, I floor, Sahakar Bhavan, “A” Block, Sahakar Nagar, Bangalore-560092 Ph:9 1-80-23620050, Fax:91-80-23625647, Website: www.chromous.com, email: info@chromous.cortg Spectrum of Innovation CHROME BIOTECH PVT. LTD, PCR 12 For Test reactions: |, Set-up Test RT reactions by edding the cor ponents in the following order: 2. Mix 5 ul of control RNA Template (1 jg) and 400ng of Reverse Primer, 300 ng of forward primer {gene specific). Add DEPC water to make the volume of the reaction mix to 23 ul, 3. Heat inactivate at 65°C for 10 min and immediately Chill on ice, & Setup Test RT PCR reactions by adding te components in the following order: Add 25 yl Reaction mix provided ‘Add 23 ul RNA- primers mix and finally add 2 ul enzyme mix. Cycle Condition: 42°C ToC [OFC aE ae [aR aC 30min] 18min [et it t tte Necks ——— > the amplicon Attings Extension snc Final extension onditions will vary depending on the size of the amplicon, N- Number of PCR cycles (vary depending on the amplicon) No.842; I floor, Sahakar Bhavan, “A” Block, Sahakar Nagar, Bangalore-560092 Ph:9 1-80-23620050, Fax:91-80-23625647, Website: wurw, ‘chromous.com, email: info@chromous.com