Tth DNA Polymerase Maximo: Datasheet
Tth DNA Polymerase Maximo: Deutsche Beschreibung
Features:
The thermostability and the reverse transcriptase (RT) activity of Tth DNA polymerase is useful in amplifying DNA from RNA templates that contain G-C-rich sequences or secondary structures since the elevated temperatures serve to denature the template RNA. Higher temperatures (in contrast to other enzymes for RT-PCR) also result in increased specificity of primer hybridization and extension. The concentration of RNA template for effective reverse transcription with Tth DNA polymerase should be higher if to compare with reverse transcription directed by Reverse Transcriptases (M-MuLV, AMV).
Applications:
- PCR and RT-PCR
- cDNA synthesis
Description:
Tth DNA Polymerase is a thermostable enzyme that replicates DNA at 74 °C and exhibits a half-life of 20 minutes at 95 °C isolated from eubacterium Thermus thermophilus strain HB8.
Tth catalyzes the polymerization of nucleotides into duplex DNA in the 5´→3´ direction in the presence of magnesium and the polymerization of nucleotides into DNA using an RNA template in the 5´→3´ direction in the presence of manganese. The enzyme has a molecular weight of 94 000 daltons as estimated from the predicted amino acid sequence and exhibits 5´→3´ exonuclease activity. Tth is recommended for use in PCR, RT-PCR, reverse transcription and primer extension reactions at elevated temperature.
Concentration: 5 u/µl
Storage Buffer:
10 mM Tris-HCl, 1 mM dithiothreitol, 0.1 mM EDTA, 300 mM KCl, 0.1% Triton X-100 (v/v)*, 50% glycerol (v/v), pH 7.5 (25°C)
Reaction Buffer:
5X RT/PCR reaction buffer: 250 mM bicine (pH 8.2, by KOH, at 25°C), 580mM KOAC, 40% Glycerol
10X PCR buffer: 100 mM Tris-HCl,(pH 8.8 at 25°C), 15 mM MgSO4, 800mM (NH4)2SO4, 0.5 mg/ml BSA, 0.5% Tween 20
Usage:
1.) One step RT PCR:
- Reverse transcription and amplification in one Tube
- Advantage: The one step One step reaction eliminates the risk of cross contaminations associated with two step RT-PCR.
2.) Two step RT PCR
3.) Standard PCR
1.) One step RT-PCR
Prepare two mastermixes 25µl each:
Mix I:
|
Component |
Volume |
final conc. |
|
dNTP Mix (40mM = 10mM each) |
1,5 µl |
300 µM |
|
sterile Water |
up to 25 µl |
|
|
forward primer |
var. |
450 µM |
|
reverse primer |
var. |
450 µM |
|
template RNA |
var. |
up to 1 µg
(in steps of 1 ng, 10 ng, 100 ng, 1 µg) |
|
Total |
25 µl |
|
Mix II:
|
Component |
Volume |
final conc. |
|
5X RT-PCR buffer |
10 µl |
1X |
|
MnCl2 (25 mM) |
5 µl |
2,5 mM |
|
Tth DNA Pol. Maximo |
0.5 - 1 µl |
2.5 - 5 units |
|
Total |
25 µl |
|
Note:
- combine Mix I and Mix II on ice and gently vortex the final mixture in a PCR-tube
- collect the mixture from the tube and start cycling immediately
Cycling: One step RT PCR:
|
Step |
Cycle |
Time |
Temperature |
|
RT-reaction |
1 |
30 min |
60-70 °C |
|
Initial denaturation |
1 |
1-3 min |
95°C |
10 Cycles:
Denaturation
Annealing 1.)
Extension
|
|
30-60 sec
30-60 sec
45 sec |
94-95°C
50-70°C
72-74°C
|
20-30 cycles 3.)
Denaturation
Annealing 1.)
Extension
|
|
30 sec
30 sec
45 sec 2.) |
94-95°C
50-70°C
72-74°C |
|
Final extension |
|
7 min |
72-74°C |
1.) temperature depends on the melting temp of the primer; approximately 5°C to 8°C below Tm of primers
2.) we recommend to add 5 sec per cycle extension
3.) depends on the copy number of the RNA
2.) Two step RT PCR
|
Component for RT-reaction |
Volume |
final conc. |
|
sterile Water |
up to 20 µl |
|
|
10x Reaction buffer Rev. Transcription |
2 µl |
1X |
|
MnCl2 |
2 µl |
0.9 mM |
|
dNTP Mix (40mM = 10mM each) |
0,4 µl |
200 µM |
|
reverse primer |
var. |
450 µM |
|
template RNA |
var. |
up to 200 ng |
|
Tth Polymerase (5µ/µl) |
0.8 µl |
4 units |
Total
for the RT reaction incubate the mixture at:
60-70°C for 10-30 min. |
20 µl
|
|
|
Components for PCR-reaction |
Volume |
final conc. |
|
sterile Water |
up to 80 µl |
|
|
10x PCR-Reaction buffer |
8 µl |
0.8X |
|
dNTP Mix (40mM = 10mM each) |
0,4 µl |
200 µM |
|
reverse primer |
var. |
450 µM |
|
EGTA, 7,5 mM |
10 µl |
0.75 nM |
|
forward primer |
var. |
150 nM |
|
Tth Polymerase (5µ/µl) |
0.8 µl |
4 units |
Total
gently vortex and add the 80 µl PCR-mastermix to the RT-PCR reaction (after incubation) at room temperature.
Total volume:
continue cycling immediatelly! see next line |
80 µl
100 µl
|
|
|
Step (PCR reaction) |
Cycle |
Time |
Temperature |
|
Initial denaturation |
1 |
1-2 min |
95°C |
10 Cycles:
Denaturation
Annealing 1.)
Extension
|
|
30-60 sec
30-60 sec
45 sec |
94-95°C
50-70°C
72-74°C
|
20-30 cycles 3.)
Denaturation
Annealing 1.)
Extension
|
|
30 sec
30 sec
45 sec 2.) |
94-95°C
50-70°C
72-74°C |
|
Final extension |
|
7 min |
72-74°C |
1.) temperature depends on the melting temp of the primer; approximately 5°C to 8°C below Tm of primers
2.) we recommend to add 5 sec per cycle extension
3.) depends on the copy number of the RNA
3.) Standard PCR
Prepare two mastermixes 50 µl each:
Mix I:
|
Component |
Volume |
final conc. |
|
dNTP Mix (40mM = 10mM each) |
200 µl |
200 µM |
|
sterile Water |
up to 50 µl |
|
|
forward primer |
var. |
400 µM |
|
reverse primer |
var. |
400 µM |
|
template RNA |
var. |
up to 0.5 µg |
|
Total |
50 µl |
|
Mix II:
|
Component |
Volume |
final conc. |
|
sterile water |
up to 50 µl |
|
|
10X PCR buffer |
10 µl |
1X |
|
Tth DNA Pol. Maximo |
0.5 - 0.8 µl |
2.5 - 4 units |
|
Total |
50 µl |
|
Note:
- combine Mix I and Mix II on ice and gently vortex the final mixture in a PCR-tube
- collect the mixture from the tube and start cycling immediately
|
Step (PCR reaction) |
Cycle |
Time |
Temperature |
|
Initial denaturation |
1 |
1-2 min |
94-95°C |
10 Cycles:
Denaturation
Annealing 1.)
Extension
|
|
30-60 sec
30-60 sec
45 sec |
94-95°C
50-70°C
72-74°C
|
20-25 cycles 3.)
Denaturation
Annealing 1.)
Extension
|
|
30 sec
30 sec
45 sec 2.) |
94-95°C
50-70°C
72-74°C |
|
Final extension |
|
7 min |
72-74°C |
1.) temperature depends on the melting temp of the primer; approximately 5°C to 8°C below Tm of primers
2.) we recommend to add 5 sec per cycle extension
3.) depends on the copy number of the RNA
Transportation: on blue ice
Storage: at -20°C for 24 months
|
