How much RNA should be used for cDNA synthesis?
Most protocols suggest to use between 0.1 to 1 ug RNA.
What is the minimum amount of cDNA for qPCR?
How much cDNA is recommended per PrimeTime qPCR Assay reaction? For optimal performance of the assays, use 1 to 100 ng cDNA per 20 µL reaction.
How much RNA do you need for PCR?
You should get at least 1ug RNA/million cells and can do RT with 100 ng RNA.
Is cDNA more stable than RNA?
cDNA is not subject to RNase degradation, making it more stable than RNA. In RT-PCR, the starting RNA is subsequently degraded, dsDNA is produced, and PCR amplification proceeds in the usual manner.
What is the minimum concentration of RNA for cDNA synthesis?
What is the minimum concentration of RNA (e.g 100ng/ul) that can be used in the applied biosystems RNA to cDNA synthesis kit? For the RNA precipitated, most concentrations are around 10ng/ul with some even lower than 10ng/ul. The 260/230 ratios are also poor, most below 1.
How is GdNA removed before making a cDNA?
The traditional method of gDNA removal is the addition of DNase I to preparations of isolated RNA. DNase I must be removed prior to cDNA synthesis since any residual enzyme would degrade single-stranded DNA.
When to use random primers for cDNA synthesis?
Use random primers for cDNA synthesis; use an RT kit with a new generation of RT conducive to synthesis at > 50C and perform RT @ > 50C for 2 hours. I have enclosed some pdf information on the merits of Life technologies superscript IV in that regard
How to maximise the yield of RNA extraction?
Also whether you are using a traditional RNA extraction column pre heat your elutant – water or TE – to 37C; add to the column; wait 5 min; spin to elute; add this eluted material back to the column; wait a further 5 min;then elute. This will maximise yield and concentration especially important for rate limiting amounts of material