Hieff^TM Blood Advanced Direct PCR Kit

Mfr.No.

10188ES20, 10188ES60

Description

The Blood Advanced Direct PCR 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 Whatman903TM and FTATM 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.

Product Components

No.	Name	10188ES20	10188ES60
10188-A	2× Hieff® Blood Advanced PCR Buffer	500 μL	2.5 mL
10188-B	Hieff® Advanced High-Fidelity DNA Polymerase Mix	20 μL	100 μL
10188-C	Positive control primer mix (10 μM each)	100 μL	200 μL
10188-D	10× DNA loading buffer	1 mL	1 mL

Product Details

Recommended PCR reaction systems.

Components	Volume（μL）
ddH2O	to 50 μL
2× Hieff® Blood Advanced PCR Buffer	25 μL
Forward Primer (10 μM)	2 μL
Reverse Primer (10 μM)	2 μL
Hieff® Advanced High-Fidelity DNA Polymerase Mix	1 μL
Blood sample	X

1. Mix well the components before use.
2. A final concentration of 0.4 μM per primer is recommended, too high will result in non-specific amplification.
3. The optimal whole blood template concentration ranges from 0.5% to 20%, and the recommended use is 10% as the initial trial condition, that is, 5 μL of whole blood is added as a template to the 50 μL reaction system, taking care to avoid aspirating blood clots. For dried blood stains stored on Whatman® filter cards, approximately 1 mm2 of round pieces of paper with blood stains can be taken and amplified directly into the PCR reaction without pretreatment.

Reaction program

Cycle step	Temp.	Time	Cycles
Initial denaturation	95℃	5 min	1.00
Denaturation	95℃	15 sec	30-35
Annealing	60℃	15 sec
Extension	72℃	3-10 sec/kb
Final extension	72℃	5 min	1.00

The annealing temperature is a generic Tm value or 1-2°C below the primer Tm value. If the amplification product is less specific, an annealing temperature gradient can be established to find optimal annealing conditions.

Extension time of 10 sec/kb can amplify most of the target fragments below 8 kb, and 3-5 sec/kb can amplify most fragments below 2 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.

Specifications

Fidelity (vs. Taq)	1 ×
Hot Start	Yes
Overhang	3'-A
Polymerase	Taq DNA Polymerase (Cat: 10726)

Applications

Direct PCR

Note	1. It is recommended that the blood template be used as 10% of the total reaction volume, that is, 5 μL of whole blood is added to the 50 μL reaction system as the template, taking care to avoid aspirating blood clots. 2. Extension time of 10 sec/kb can amplify most of the target fragments below 8 KB, and 3-5 sec/kb can amplify most of the fragments below 2 KB. If the amplification efficiency is low, the time can be extended to 30 sec/kb. 3. After the PCR reaction, it is recommended to centrifuge the reaction product at 4000 rpm (1000× g) for 1-3 min to precipitate blood cell fragments, and take the supeatant for downstream analysis. 4. This product cannot be directly used for medical diagnosis. 5. For your safety and health, please wear a lab coat and wear disposable gloves.
Storage	The products should be stored at -15℃ ~ -25℃ for 1 year

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Combination SKU	Supplier No.	Size	Your price	Quantity
BHR20800020-1	10188ES20	20T	$25		Add to Cart
BHR20800020-2	10188ES60	100T	$105		Add to Cart

Hieff^TM Blood Advanced Direct PCR Kit

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