Phi(ɸ)Script™ 1st strand cDNA Mastermix is extremely user friendly while making cDNA. The mastermix (5X) is uniquely blended with RNase Inhibitors, dNTPs, Mg+2, Reaction buffers, oligo(dT) , random primers and Stabilisers. Our flagship enzyme PhiScript reverse transcriptase is provided in a separate vial. t’s an engineered form of Moloney Murine Lukemia Virus reverse transcriptase (M-MuLV-RT). Point mutations are introduced in ɸScript reverse transcriptase so as to reduce RNAse H Activity and to improve its thermal stability.
RNA upto 2µg is supported per reaction
Hig yield cDNA synthesis just under 30 minutes.
Efficient reverse Transcription upto 60℃
2 Step procedure. Just add your RNA and RT. Thats it !
High tolerance to RNAses.
Mastermix is optimised for reverse transcription upto 2KB.
Accurate results. Simple. Incredible prices.
Ultrafast delivery across India. Less than 3 hrs for Namma Bengaluru.
Source
PhiScript™ reverse transcriptase was cloned from MML Virus gene and expressed in E.Coli.
Total human RNA was isolated from skeletal cells. Exon junction primers were designed for human integrin mRNA transcript.
cDNA Synthesis conditions
Total RNA template (1µL=10ng), cDNA 5X mastermix(4µL each), ɸScript Reverse Transcriptase, 1µL) and make upto 20µL using Nuclease Free Water.
Cycling conditions
25℃ (5min)
42℃ (20min)– Reverse Transcription
85℃ (10min) — Inactivates RT
-20℃ — Storage
PCR Conditions
cDNA from the condition was tested and mastermix ability was checked for 3 different sizes of the same human integrin transcript ( 150bp, 650bp and 1.6kb). Appropriate forwad and reverse primers were used.
General PCR conditions:
cDNA: 2µL, For/Rev primers: 1µL each, DX/DT Extremo mastermix: 25µL and make up 50µL using nuclease free water.
PCR Cycles:
95℃ – 2min, [95℃-30s, 60℃-30s, 72℃-1min/kb]30cycles, Final extension, 72℃ for 2min.
5µL of the sample was loaded on 1.5% agarose gel and run for 30 minutes.
Reverse transcription temperature is crucial for certain application where RNA molecule is complex and contains high amounts secondary structure. cDNA synthesis with these samples are always challenging. But worry not. Phiscript (ɸ) reverse transcriptase has exceptional stability to work at higher temperatures. This is also one of the critical parameters during one step reverse transcription where non-specific amplification has to be reduced.
Total human RNA was isolated from skeletal cells. Exon junction primers were designed for human integrin mRNA transcript.
cDNA Synthesis conditions
Total RNA template (1µL=10ng), cDNA 5X mastermix(4µL each), ɸScript Reverse Transcriptase, 1µL) and make upto 20µL using Nuclease Free Water.
Cycling conditions
25℃ (5min)
37℃ (30min), 42℃ (30min), 50℃ (30min), 60℃ (15min) — Reverse Transcription (Separate vials)
85℃ (10min) — Inactivates RT
-20℃ — Storage
PCR Conditions
cDNA from the condition was tested and mastermix ability was checked for 2 different sizes of the same human integrin transcript (150bp and 1.6kb). Appropriate forwad and reverse primers were used.
General PCR conditions:
cDNA: 2µL, For/Rev primers: 1µL each, DX/DT Extremo mastermix: 25µL and make up 50µL using nuclease free water.
PCR Cycles:
95℃ – 2min, [95℃-30s, 60℃-30s, 72℃-1min/kb]30cycles, Final extension, 72℃ for 2min.
5µL of the sample was loaded on 1.5% agarose gel and run for 30 minutes.
DX/DT cDNA 5X performance was compared against other brands in the market. Total human RNA was isolated from skeletal cells. Exon junction primers were designed for human integrin mRNA transcript.
cDNA Synthesis conditions
Total RNA template (1µL=10ng), cDNA 5X mastermix(4µL each), ɸScript Reverse Transcriptase, 1µL) and make upto 20µL using Nuclease Free Water.
Cycling conditions
25℃ (5min)
42℃ (30min) — Reverse Transcription (Separate vials for competitors as per their instruction)
85℃ (10min) — Inactivates RT
-20℃ — Storage
PCR Conditions
cDNA from the condition was tested and mastermix ability was checked for reverse transcription of 650bp fragment. Appropriate forwad and reverse primers were used.
General PCR conditions:
cDNA: 2µL, For/Rev primers: 1µL each, DX/DT LEO PRIME Mastermix: 25µL and make up 50µL using nuclease free water.
PCR Cycles:
95℃ – 2min, [95℃-30s, 60℃-30s, 72℃-1min/kb]30cycles, Final extension, 72℃ for 2min.
5µL of the sample was loaded on 1.5% agarose gel and run for 30 minutes.
Long time storage: -20℃, 2 years
Day to day usage: 4℃, One Month
Always prevent the master-mix from primer and nucleases contamination.
Protocol for this product can be found here.
All the products are for research purpose only. Not for human diagnostics.
