Mighty Leo Mastermix is 2X concentrated solution for PCR reaction which contains dNTPs, Taq DNA Polymerase, Mg+2 and other critical reaction components. It doesn’t contain primers and DNA template. Leo Mastermix is designed for routine PCR and also for amplification of high GC templates. The pre loaded green dye makes it easier for users to load directly on the agarose gel post PCR.
Leo mastermix is best suited for high GC targets.
PCR Amplifications upto 5KB is supported.
Also suited for simple and routine PCR applications
Making genotyping simpler and reliable.
High tolerance for many PCR inhibitors/reagents.
Preloaded with green dye for direct gel loading.
Making Colony PCR extremely simple, accurate and fast.
Ultrafast delivery! 2-3 working days for India and 2 hrs for Namma Bengaluru.
Features
Source
The mastermix contains Taq DNA Polymerase whose gene was cloned from Thermus aquaticus and expressed in E.Coli.
1ng of Lambda (λ) phage DNA was taken for amplification. 500bp , 3kb and 5kb fragments were amplified using LEO Mastermix. Average GC content: 57%
PCR Conditions
Lambda template (1µL), forward & reverse primers (1µL each), LEO Mastermix (25µL) and make upto 50µL using Nuclease Free Water.
Cycling conditions and Gel
95℃ (2min), 25 Cycles [ 95℃-30s; 58℃-30s; 72℃-1min/kb], 72℃- 5to 10 minutes. 5µL of sample was loaded directly in 1% Agarose Gel which was run just for 15 minutes.
Leo Mastermix has extreme capabilities to detect positive colonies. To demonstrate this, two plasmids were chosen.
Plasmid 1: 6.7 KB total length. Gene fragment: 1.6KB
Plasmid 2: 8KB total length. Gene fragment: 2.9KB
PCR Conditions
One single colony was picked using 10µL tip and suspended in 100µL of nuclease free water. From this suspension, 1µL, 3µL and 1µL(diluted 10 times) were taken for colony PCR.
Forward & Reverse(10µM): 1µL each
Leo Mastermix: 25µL
Final Volume: 50µL
Cycling conditions and Gel
Plasmid 1: 95℃ (2min), 25 Cycles [ 95℃-30s; 58℃-30s; 72℃-1.5min], 72℃- 2 minutes.
Plasmid 2: 95℃ (2min), 25 Cycles [ 95℃-30s; 58℃-30s; 72℃-3min], 72℃- 5 minutes.
10 µL of sample was loaded directly in 1% Agarose Gel which was run just for 30 minutes.
One of the most critical feature for any mastermix is its ability to withstand ‘Freeze-Thaw’ cycles. Leo™ mastermix out-performs this test. It’s astonishingly stable for at least 50 freeze thaw cycles tested.
Freeze thaw test
Leo™ mastermix was taken out from -20℃ freezer and thawed in hand for 3-4 minutes. After this, samples were vortexed and spun down for 10 mins. It was then kept on ice for 30 mins (for PCR activities),later were stored in -20℃ for 90 mins. Amplification was tested for every 15, 30, 40 and 50 cycles.
PCR Conditions
Sample: Lambda DNA (1ng), target 300bp
Cycling conditions and Gel
95℃ (2min), 25 Cycles [ 95℃-30s; 58℃-30s; 72℃-30s], 72℃- 2 minutes.
10 µL of sample was loaded directly in 1% Agarose Gel which was run just for 30 minutes.
At dx/dt, we believe in providing high quality mastermixes at an affordable price. This would not be possible with cutting col-chain costs. Hence, we designed an unique buffer which keeps Leo™ mastermix and few other mastermixes stable at room temperature. At least for a week !
Leo Mastermix was kept in hot air oven set at 45℃ (Really!). It was stored for 8 days.
PCR Conditions
Sample: Lambda DNA (1ng), target 300bp fragment
Cycling conditions and Gel
95℃ (2min), 25 Cycles [ 95℃-30s; 58℃-30s; 72℃-30s], 72℃- 2 minutes.
10 µL of sample was loaded directly in 1% Agarose Gel which was run just for 30 minutes.
Leo mastermix contains unique buffer and reaction mix which can tolerate humic acid/fulvic acid which may be present in your DNA template samples. Humic/fulvic acid are PCR inhibitors and severely impact Taq DNA polymerase performance.
Study Details
Humic acid/Fulvic acid salt (70:15) was dissolved in nuclease free water and stock was prepared. To a PCR reaction volume of 50μL, concentration of humic acid/fulvic acid salt was tested at 3.5ng/μL, 7ng/μL, 10ng/μL, & 20ng/μL. A competitor product (Ampliqon) was also tested for comparison.
PCR Conditions
Sample: Lambda DNA (100pg), target 1KB fragment, forward/reverse primers 1μL each
Cycling conditions and Gel
95°C (2min), 25 Cycles [ 95°C-30s; 58°C-30s; 72°C-1min/kb], 72°C- 2 minutes. 10 μL of sample was loaded directly in 1% Agarose Gel which was run just 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.
