After cDNA preparation, I need to amplify this cDNA for my interest of gene so what is the concentration range (amount of cDNA in terms of either ng or ug) during PCR amplification. From some source I got (1ng- 100ng) should use during gene specific amplification. How this range affects PCR?
what is the relation between total reaction volume and concentration of cDNA/DNA during PCR?
Do you know how much RNA you added to your mix to make the cDNA?
On our lab we standardized all cDNA making. All reaction are done with 1 ug of RNA. The final product, the cDNA, is diluted to 125 ul. For RT-qPCR we use 2,5 ul of cDNA in an 25 ul reactie. When we would like to perform a PCR of which the product is separated on gel, for fusion genes, we use 1 ul cDNA. This can be increased depending on how many copies are expected to be present in the sample.
The quality of your cDNA and the synthesis of the cDNA is depending on the quality of your RNA. This makes controle genes and samples important for understanding your results. Always use a housekeeping gen and standaard reference samples to compare measurements/experiments.
Dear Jain,
If you follow the hand book/ guidelines provided with the respective polymerase enzyme, you will get the suitable range of template amount for the enzyme. For example, you are using a polymerase whose template limit is ≤1 µg/reaction (reaction volume = 100 µl). If you want to go for 20 µl of reaction volume, you have to take ≤200 ng of your template (cDNA in your case). For cDNA concentration, you can assume a linear relationship with the total RNA template amount used for cDNA synthesis. Suppose, you have taken 400 ng of total RNA to make cDNA in 20 µl reaction volume. Assume all the RNAs converted into cDNA. So, theoretically 400 ng of cDNA is present in 20 µl of reaction volume. So, the concentration of cDNA is 20 ng/ µl. Now, for your 20 µl PCR reaction volume you have to add maximum 2 µl (10 % template volume of total reaction volume) of cDNA, which will cover the polymerase range.( 2 µl of cDNA contain total 40 ng of cDNA). Do not go for the higher limit of template amount. Excess template inhibits the reaction. For me, I use 10 times diluted cDNA for PCR reaction (per 20 µl of reaction volume, total template becomes 2 - 2.5 ng) and gives good result.
Hope it will help.
maximum in a 25microliter reaction use a max vol of cDNA of 5microliter
Debaprasad koner thanks, I do agree with you, but in Taq pol and Q5 poly protocol, there is only gDNA and plasmid and viral DNA concentration was mentioned and nothing is about the concentration range for cDNA.
I want to know about concentration range of cDNA for Taq pol and q5 pol ?
Dear Jain,
Easily you can follow the concentration range mentioned for gDNA. Chemically the thing is same. Polymerase does not know the origin of template. Its better to use diluted cDNA (less amount of template) to get a good result.
Hope for the best.
It's difficult to know how much cDNA was made in reverse transcription reaction, because of the presence of the template RNA and nucleotides. So most people add an amount of cDNA corresponding the amount of RNA that they added to the reverse transcription. And this amount could be anywhere in the 1 ng to 100 ng range, with 25-50 ng being a commonly used amount. There is also some dependence on the expression level of the gene that you are interested in. If it is very highly expressed, then it will be a large proportion of the cDNA and you would need less to detect it. If it is a very rare transcript, you might need to use more cDNA and/or to run multiple reactions to detect it.
regardless of concentration, dilute that cDNA sample at least 3x or else the cDNA reaction reagents will inhibit downstream PCR applications
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