Description
Product description
This Kit is a kit that enables direct PCR amplification of whole blood samples without DNA purification or sample pretreatment. This kit is compatible with fresh blood containing conventional anticoagulants such as EDTA, heparin, citrate, frozen blood and commercial Whatman903™ and FTA™ dried blood stains. The kit contains a combination of genetically engineered DNA Polymerase with high fidelity and tolerance to PCR inhibitors. It can efficiently amplify 8 kb genomic fragments under rapid elongation conditions, for the fragments less than 2 kb, the extension time of 3-5 sec/kb can be set to complete the amplification, thus significantly reducing the identification and detection time of blood samples.
Advanced High-Fidelity DNA Polymerase Mix has a flat 3 'end of the amplified product, which is suitable for one-step rapid cloning (CAT#N312081) and TOPO cloning.
The Positive control primer mix (10 μmol/L each) provided in the kit is capable of amplifying a fragment 237 bp in length from the upstream conserved sequence of the sox21 gene in mammals and most vertebrates, which could be used as a positive control.
Components
|
Components No.
|
Name
|
N132022E
(50 T)
|
N132022S
(100 T)
|
|
N132022-A
|
2× Blood Advanced PCR Buffer
|
1.25 mL
|
2.5 mL
|
|
N132022-B
|
Advanced High-Fidelity DNA Polymerase Mix
|
50 μL
|
100 μL
|
|
N132022-C
|
Positive control primer mix (10 mol/L each)
|
100 μL
|
200 μL
|
|
N132022-D
|
10×DNA loading buffer
|
1 mL
|
1 mL
|
Shipping and Storage
The product should be stored at -25 ~ -15℃ for one year. Please avoid repeated freeze-thaw.
Notes
1. The recommended amount of blood template is 10% of the total reaction volume, i.e. 5 μL of whole blood in 50 μL reaction system as a template, taking care to avoid suction of blood clots.
2. Most of the target fragments below 8 kb could be amplified with the extension time of 10 sec/kb, and most of the fragments below 2 kb could be amplified with the extension time of 3-5 sec/kb. If the amplification efficiency is low, it can be used when extended to 30 sec/kb.
3. After PCR reaction, it is recommended to centrifuge the reaction product at 4000 rpm (1000 x g) for 1-3 min to precipitate blood cell debris and remove the supernatant for downstream analysis.
4. This product should not be used directly for medical diagnosis.
5. For your safety and health, please wear lab coats and disposable gloves for operation.
6. This product is for research use ONLY!
Instructions
1. Reaction System
Table 1 Amplification protocol
|
Components
|
Volume (μL)
|
|
2× Blood Advanced PCR Buffer
|
25
|
|
Forward Primer (10 μmol/L)
|
2
|
|
Reverse Primer (10 μmol/L)
|
2
|
|
Advanced High-Fidelity DNA Polymerase Mix
|
1
|
|
ddH2O
|
to 50
|
【Note】: *All components should be thoroughly mixed before use.
1) Primer final concentration: The recommended final concentration for each primer is 0.4 μmol/L, too high will result in non-specific amplification.
2) Template usage: The optimal whole blood template concentration range is 0.5%-20%, and the recommended dosage is 10% as the initial trial condition, i.e. 5 μL whole blood in 50 μL reaction system as a template, taking care to avoid suction of blood clots. For dried blood stains stored on WhatmanTM filter paper cards, a round paper of about 1 mm2 with blood stains can be used for amplification without pretreatment and directly placed into the PCR reaction solution.
2. Reaction Program
|
Cycle steps
|
Temperature (℃)
|
Time
|
Cycles
|
|
Predenaturation
|
95
|
5 min
|
1
|
|
Denaturation
|
95
|
15 sec
|
30-35
|
|
Annealing
|
60
|
15 sec
|
|
Extension
|
72
|
3-10 sec/kb
|
|
Final extension
|
72
|
5 min
|
1
|
【Note】:
a. Annealing temperature: please refer to the theoretical Tm value of the primer or 1-2°C below the primer Tm value. If amplification product specificity is poor, an annealing temperature gradient can be established to find optimal annealing conditions.
b. Extension time: Most of the target fragments below 8 kb could be amplified with the extension time of 10 sec/kb, and most of the fragments below 2 kb could be amplified with the extension time of 3-5 sec/kb. If the amplification efficiency is low, the time can be appropriately extended to 20-30 sec/kb and should not exceed 60 sec/kb.
