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Direct PCR Lyophilisate

lyophilised Master Mix for direct PCR amplification from blood, animal and plant tissue

Cat. No. Amount Price (EUR) Buy / Note
PCR-160S 192 reactions x 20 μl 205,00 Add to Basket/Quote Add to Notepad
PCR-160L 960 reactions x 20 μl 820,00 Add to Basket/Quote Add to Notepad
Example: Direct Amplification from blood, saliva and plant tissues (Direct PCR was used to amplify a 295 bp long fragment of the beta-actin gene from whole blood and saliva and a 550 bp fragment of the Leucin tRNA gene from grass leaves and wheat seeds.)
Example: Direct Amplification from blood, saliva and plant tissues (Direct PCR was used to amplify a 295 bp long fragment of the beta-actin gene from whole blood and saliva and a 550 bp fragment of the Leucin tRNA gene from grass leaves and wheat seeds.)

For in vitro use only!

Shipping: shipped at ambient temperature

Storage Conditions: store at ambient temperature
Store in an aluminium-coated bag or on a dry place.
Lyophilisates may hydrate at humidity levels >70 % when sealing is opened.

Shelf Life: 12 months

Applications:

  • Direct PCR amplification of target DNA without any prior DNA purification step from various sample types such as whole blood, saliva, mouse tissues (tail, heart, liver, large intestine, small intestine, kidney, stomach, ear, brain, spleen), zebra fish fin, pork, beef and plant tissues (leaf and seed)
  • Allele-specific PCR
  • PCR for genotyping
  • PCR for selection of genetically modified organisms (GMO)

Description:
Direct PCR Lyophilisate is delivered in PCR reaction tube strips or 96-well plates preloaded with a complete master mix in a dry, room temperature stable format. The lyophilisate combines highest performance with convenience of use and stability. There is no need for freezing, thawing or pipetting on ice. The few remaining pipetting steps minimize the risk of errors or contaminations.
To perform PCR, fill up the vials with primers, Additives Mix and PCR-grade water and add DNA template. If necessary, centrifuge to remove bubbles, vortex the vials to assure homogeneity and start cycling.
The lyophilisate is designed for PCR amplification directly from whole blood, animal tissues and plant tissues without the need of prior DNA purification processes.
The robust enzyme in combination with a specially optimized unique buffer system resists various PCR inhibitors of blood and tissue samples.
The enzyme lacks a 3'→5' exonuclease activity making this kit an ideal choice for allele specific PCR which is routinely used for various genotyping applications.

Content:
Direct PCR Lyophilisate
Preloaded lyophilisates of Direct PCR Mastermix containing Hot Start Taq polymerase, nucleotides, optimized reaction buffer, stabilizers

Additives Mix (purple cap)
2 x conc.

DNA Extraction Buffer (yellow cap)
10 x conc.

PCR grade water (white cap)


Sample Preparation
Whole blood or saliva

  • Add directly 1-2 μl of sample to the PCR reaction mix without any pre-treatment.
    Heparin, EDTA or citrate treated whole blood is suitable for this kit.


Animal or Plant tissue

  • Prepare 1x DNA Extraction Buffer (yellow cap) by diluting the provided 10 x conc. Buffer with PCR-grade Water. Aliquot 20 μl of the 1x DNA Extraction Buffer into a 1.5-2 ml microtube.
  • Take a small piece of tissue (about 2-3 mm in diameter) from animal or plant tissue. Plant seeds should be cracked down to a size of less than 1 mm diameter in a mortar, bead beater or tissuelyser.
  • Add the tissue sample into the 1x DNA Extraction Buffer containing tube.
  • Briefly mix by tapping or vortexing.
  • Incubate for 3 min at RT to allow tissue lysis and DNA releasing.
  • Centrifuge briefly.
  • Transfer 1-2 μl of the lysate supernatant into the PCR reaction mix.
    The lysate supernatant will be stable for several weeks if stored at -20°C.

Recommended PCR assay:
The preparation of a primer mix is recommended to minimize pipetting errors.

Comp.capstock conc.final conc.20 μl assay
Direct PCR Lyophilisate 1 x1 tube
Additives Mixpurple2 x1 x10 μl
forward Primer1) 10 μM400 nM0.8 μl
reverse Primer1) 10 μM400 nM0.8 μl
Sample Preparation 1-2 μl
PCR-grade waterwhite fill up to 20 μl


Recommended cycling conditions:
Before cycling, vortex PCR tubes or plates to assure homogeneity and centrifuge briefly to remove possible bubbles.
Initial
denaturation
95 °C5 min1x
Denaturation95 °C20 sec35-40x
Annealing1)45-68 °C30 sec35-40x
Elongation2)72 °C30 sec - 4 min35-40x
Final elongation72 °C2 min1x

1) The annealing temperature depends on the melting temperature of the primers used.
2) The elongation time depends on the length of the fragments to be amplified. A time of 1 min/kb is recommended.