Internal Amplification control in PCR

[dgadiyar]

Kindly let me know how to design an internal amplification control for detection of hil A gene of Salmonella in stool samples. this is mainly to rule out PCR inhibitors in the DNA extracted from stool

thanks and regards

dgadiyar

[danfive]

Spiked controls can be used to rule out PCR inhibitors. If you make a set of PCR reactions and add sample DNA template then spike with your positive control, you should get amplification in all reaction tubes, unless PCR inhibitors are present.

[Agustin Franco]

Hi There:

The problem with your sugestion is that by adding an internal control on the DNA solution in question may tell you whether you have inhbitors or not, but it does not tell you whether your extraction was successful and in fact a positive sample may be negative,of course, in absence of inhibitors!

[Aga]

I usually solve this problem by dealing with all the samples in the same way beginning with extraction. You can spike your samples with specific amount of your bacterial cells as a positive control and perform DNA extraction and all further procedures as usual.

[Anastasia_S]

Im a novice in PCR, but in my field amplification of some housekeeping gene along with your gene of interest is used as an internal control (so you'll have at least 2 sets of primers)

[amicia]

The best way is to clone a know fragment and put it into a plasmid. You can add the plasmid at the begging of extraction. At the moment of PCR reaction, you have to do a duplex with two pair primers: one for your target gene and the other to amplify the fragment into the plasmid. If the plasmid frgament PCR is negative, you have an inhibition (or the extraction is failed), in case you have an inhibition try with a ten fold DNA diluition.

[Agustin Franco]

Originally Posted by dgadiyar

Kindly let me know how to design an internal amplification control for detection of hil A gene of Salmonella in stool samples. this is mainly to rule out PCR inhibitors in the DNA extracted from stool

thanks and regards

dgadiyar

Hi there:

one does not have to reinvent the wheel to get an internal control
The best way to do this is to find any article which shows a good primer set for the detection of the human beta actin gene. The human beta actin fragment should coamplify with your Salmonella genes.
for Amicia: the problem with plasmids is that because of their circularity, and copy number, this form of DNA can withstand more abuse than chromosomal DNA and these are detected much more easily than the original DNA target presumptively of chromosomal origin.