One.Direct.Step RT-qPCR Kit for Probes (without ROX; with high-ROX)

Kit content:

Extraction Buffer: 1x concentrated

Direct Enzyme: Mix of engineered reverse transcriptase, mAB-inhibited hot start polymerase, dNTPs, reaction buffers, enhancers and additives
PCR-grade Water

Optional: ROX Dye

Easy Preparation (e.g. 50 µl reaction volume)

1. Whole Blood (not treated heparin-, EDTA- or citrate-treated whole blood)

• Add 2-5 µl whole blood (for 50 µl reaction volume)
 without any pre-treatment directly to the RT-PCR assay.

2. Swab Samples from nasal or swaps

• fill 200 µl Extraction Buffer in a 1,5 ml Tube

• Cut the tip with nasal or throat swap and put to micro
  tube and vortex about 15-20 sec

• let absorb and incubate at room temperature for about
  3 min

• press the tip of the swap to the wall of the microtube
  and take it out

• centrifuge the tube extensively and transfer, for a 50µl
 reaction volume, 2-5 µl of the supernatant to your RT-
 PCR assay.

3. Animal or Plant Tissue samples

• Prepare a small piece from animal or plant tissue not
  exceeding 8 mm in diameter.

• Crack plant seeds to less than 1 mm in diameter using
  a cell-disrupter, Tissue-lyser or small hammer.

• Add 1x Extraction Buffer to the tissue sample using:
 

Sample size

1-2 mm - 50 µl

3-4 mm - 100 µl

5-8 mm - 200 µl

• mix Extraction Buffer and sample briefly and
  incubate for about 3 min at room temperature

• Centrifuge extensively and and transfer 2-5 µl (for
  50 µl reaction volume) of the supernatant to your
  RT-qPCR assay

 
Preparation of the RT-PCR Assay

Add the following components to a nuclease-free microtube. Pipette on ice and mix the components by pipetting gently up and down.

1.) for each PCR-Target (Multiplex PCR) take Xn+1 primers and the related amount of TaqMan probe

Note: For each primer on have to optimize the best assay parameters. The optimal primer can vary from 100 - 500 nM.

Reverse transcription and thermal cycling:
Place the vials into a real-time PCR cycler and start the following program.
 

Protocol for standard PCR cycler combined with gel - based DNA analysis the following cycling protocol is recommended:

2.) 10 min for  amplicons < 200 bp; each 100 bp fragment length need about 3 min longer incubation time

3.)The annealing temperature depends on the melting temperature of the primers.
^4.) The elongation time depends on the length of the amplicon. A time of 1 min for a fragment of 1,000 bp is recommended.

Note:
For optimal specificity and amplification an individual optimization of the recommended parameters may be necessary. Note that optimal reaction times and temperatures should be adjusted for each particular sample/primer pair.