genesig Human Y Chromosome Quantification Kit by Real-time PCR

Kits and Assays / Kits-PCR, genesig™

-20°C

This kit provides reagents for the detection and quantification of human Y-chromosome. The primers detect a region of Y chromosome in the TSPY1 gene that is not present in the X-chromosome. The amplicon is present at multiple copies within the Y chromosome which increases the sensitivity for detecting very small amount of genomic DNA and even degraded DNA.

The kit contains a number of additional components to avoid both false positive and false negative test results.

Sensitivity:
25pg of target DNA

Real-time PCR
When resuspended, this kit provides primers that have been tested for priming specificity, and amplification efficiency at optimal concentrations. Real-time PCR is a very sensitive technology and it is not recommended to use more or less than the specified amount of primer and probe in each reaction. However, final reaction volumes between 15ul and 50ul are often successful and maybe tested at the user’s discretion.

Kit Components:
Human Y-chromosome primer/probe mix (150 reactions, brown)
Positive control genomic DNA template (for standard curve, red)
Internal extraction control primer/probe mix (150 reactions, brown)
Internal extraction control DNA (150 reactions, blue)
RNAse/DNAse free water (white) for resuspension of primer/probe mixes and internal extraction control DNA
Template preparation buffer (yellow) for resuspension of positive control template and standard curve preparation

Kit Storage and Stability:
This kit is stable at room temperature but should be stored at -20°C on arrival. We do not recommend using the kit after the expiry date stated on the pack. Once the lyophilized components have been re-suspended, unnecessary repeated freeze/thawing should be avoided. The kit is stable for six months from the date of resuspension under these circumstances.
If a standard curve dilution series is prepared this can be stored frozen for an extended period. If you see any degradation in this serial dilution a fresh standard curve can be prepared from the positive control.

Positive Control:
For the purpose of DNA quantification, and as a positive control for the PCR set up, the kit contains positive control genomic DNA. This can be used to generate a standard curve of Y-chromosome concentration. Alternatively the positive control can be used at a single dilution for a qualitative analysis of the samples. Each time the kit is used, at least one positive control reaction must be included on the run. A positive result indicates that the primers and probes for quantification of the Y-chromosome are working on that particular run. If a negative result is obtained the test results are invalid and must be repeated. Care should be taken to ensure that the positive control does not contaminate any other kit component which would lead to false positive results. This can be achieved by handling this component in a Post PCR environment.

Negative Control:
To confirm absence of contamination, a negative control reaction should be included every time the kit is used. For this reaction, the RNAse/DNAse free water should be used instead of template. A negative result indicates that the reagents have not become contaminated. If a positive result is obtained the results should be ignored and the test samples repeated. Possible sources or contamination should first be explored and removed.

Internal DNA Extraction Control:
When performing DNA extraction, it is often advantageous to have an exogenous source of DNA template that is spiked into the lysis buffer. This control DNA is then co-purified with the sample genomic DNA and can be detected as a positive control for the extraction process. Successful co-purification and real-time PCR for the control DNA also indicates that PCR inhibitors are not present at a high concentration. A separate primer and probe mix are supplied with this kit to detect the exogenous DNA using real-time PCR. The primers are present at PCR limiting concentrations which allows multiplexing with the target sequence primers. Amplification of the control DNA does not interfere with detection of the Y-chromosome target DNA even when present at low copy number. The Internal control is detected through the VIC channel and gives a CT value of 26+/-3.

Reagents and Equipment To Be Supplied By The User:
1. Real-Time PCR Instrument
2. DNA extraction kit
This kit is recommended for use with genesig EASY DNA/RNA Extraction kit. However, it is designed to work well with all processes that yield high quality DNA with minimal PCR inhibitors.
3. PrecisionPLUSTM or PrecisionFASTTM 2x qPCR Mastermix
This kit is designed to work well with all commercially available Mastermixes. However, we recommend the use of Primerdesign 2x PrecisionPLUSTM or PrecisionFASTTM Mastermix.
4. Pipettors and Tips
5. Vortex and centrifuge
6. Thin walled 0.2 ml PCR reaction tubes

Suitable Sample Type:
All kinds of sample material suited for PCR amplification can be used. Please ensure the samples are suitable in terms of purity, concentration and DNA integrity. An internal PCR control is supplied to test for non specific PCR inhibitors. Always run at least one negative control with the samples. To prepare a negative control, replace the test sample with RNAse/DNAse free water.

PRODUCT MANUAL:
http://www.primerdesign.co.uk/assets/files/human_y_chromosome_dna_quantification.pdf?timestamp=1480694414