[Debian-olpc-devel] Quality control solutions
no-reply at tataa.com
Sun Sep 4 12:43:10 UTC 2011
Quality control solutions
Eliminate problems with contaminating DNA
PCR can amplify extraordinary small amounts of RNA and DNA. The high sensitivity of these methods makes them sensitive to contaminating DNA. The contaminating DNA can be
genomic DNA from impure RNA preps
carryover DNA from previous PCR products
traces of DNA from the organism used to produce the PCR enzymes
To trust your results and ensure quality you must take action to prevent contamination problems. We would like to share our best tips:
How do I control if I have contaminating gDNA in my qPCR without spending all my money on RT(-)/NoRT controls?
To cost efficiently approach the problem of gDNA contamination we suggest ValidPrime. ValidPrime is a system to measure the contribution from gDNA to the Cqs in RT(+)-PCR using proprietary gDNA specific assay ignorant to cDNA, and testing the sensitivity of gene specific qPCR on a gDNA standard. The system is more sensitive than traditional RT(-)-qPCR controls (NoRTs), and you can save a lot of control reactions by using the ValidPrime approach (see table here). Just add the ValidPrime assay to the list of assays, and the gDNA control to the list of samples, and run. ValidPrime will minimize the amount of control reactions and hence your costs, as well as your efforts.
If I do have contaminating DNA in my qPCR, how do I solve that problem?
When contaminating DNA contribution to the qPCR signal is too high (can be measured with ValidPrime) we suggest proprietary heat labile double strand specific nuclease (HL-dsDNase). Contaminating DNA is efficiently removed with the dsDNase, without any need for column purification, while primers are left intact. The dsDNase is irreversibly denatured upon heating during the PCR and will not degrade any new produced PCR products, which make the product available for (high-resolved) melt curve analysis and post PCR processing. The HL-dsDNase can also easily be incorporated in a one-step RT-PCR. The great improvement achieved using HL-dsDNase is easily verified with ValidPrime (Problem/Solution 2) and the control can be used in combination with Cod UNG (Problem/Solution 3).
In addition to setting up the PCR lab correctly, how can I prevent getting a carryover contamination in my RT-PCR from a previous PCR?
Including Uracil-DNA Glycosylase (UNG) and dUTP in all PCRs help prevent carryover contamination. Although this technique is quite common, we want to recommend using Cod Uracil-DNA Glycosylase (Cod UNG). This UNG enzyme from Atlantic cod is the only commercially available UNG enzyme that is completely and irreversibly inactivated by moderate heat treatment. The problem with most UNG enzymes is that even if they are inactivated, they will reactivate after some time in the fridge, degrade your PCR product, and giving you no chance to do downstream applications such as sequencing, cloning etc of the target. If you use Cod UNG you don't need to worry about deactivation and reactivation. Since Cod UNG is very efficient at low temperature and also rapidly inactivated at 50°C, UNG preincubation can be done at a temperature low enough to minimize cDNA generation during this step. These properties combined makes contamination control feasible in RT-PCR with no loss of sensitivity.
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