hPSC Genetic Analysis Kit Technical Tips & Tricks
- Document # 27130
- Version 1.0.0
- Jun 2018
This document contains tips and tricks for optimal use of the hPSC Genetic Analysis Kit. This document should be used in conjunction with the Product Information Sheet (PIS; Document #DX22330).
When Using the Kit for the First Time
- Read the PIS thoroughly before resuspending the Genetic Assays or preparing the qPCR Master Mix + Dye.
- The first run should be carried out on the Genomic DNA Control plus 1 - 2 samples at most. Once you are familiar with the technique and have low replicate variability, the sample number can be increased.
- Plan your plate layout in advance and have it in front of you for reference when pipetting the reactions.
Vortex, Vortex, Vortex!
- When working with DNA there is often a hesitation about vortexing, since it may lead to shearing. Although we do not recommend vortexing the stock genomic DNA samples, it is important to thoroughly vortex the DNA and Master Mix + Dye solution (prepared in step C5) for 5 seconds. This will help to reduce the variability between technical replicates.
- Likewise, the Genetic Assay and water solution prepared in step D3 should be sufficiently mixed by vortexing (5 seconds per sample). This will help to reduce the variability between technical replicates.
Genomic DNA Sample Preparation
- The amount of genomic DNA per reaction can be increased; in some cases this can improve reproducibility between replicates. e.g. Instead of using 290 ng in 58 μL (step C.4), use 580 ng or 870 ng in 58 μL.
- Mix the genomic DNA and Master Mix + Dye, prepared by the end of C5, by vortexing thoroughly (5 seconds per sample) immediately before pipetting into the plate.
- Determine the concentration and quality of genomic DNA samples using an appropriate method. Genomic DNA samples should have absorbance ratios in the range of A260/280 ~1.8 - 2.0 and A260/230 ~1.9-2.2.
- If the concentration of the sample DNA is low (< 5 ng/μL), it is beneficial to concentrate using ethanol precipitation or other suitable method prior to analysis with the hPSC Genetic Analysis Kit. Running very low concentrations of DNA may introduce high variability between replicates and lead to difficult-to-interpret results.
- The lyophilized primer-probe pellet may have become dislodged during shipping, so it is important to centrifuge the tube prior to reconstitution. If possible, try to locate the pellet and pipette the TE Resuspension Buffer directly onto the pellet for resuspension.
- It is important to mix the Genetic Assay thoroughly before use, particularly if the suspension is going to be aliquoted. This can be done by flicking the tube followed by brief centrifugation.
- Take care when pipetting the Genetic Assays; always use a clean pipette tip when moving between stocks or diluted assays to avoid cross-contamination of primer-probes.
qPCR Reaction Fails/No Amplification
All reactions fail to amplify:
Individual reactions fail to amplify:
High Variability Between Technical Replicates :