Designing the primers for qRT PCR - Query - (Sep/05/2008 )
QUOTE (HomeBrew @ Sep 6 2008, 07:16 PM)
If you design primers such that each spans two exons (say a forward primer spanning the end of the first exon and and the start of the second exon, thus eeeeEEEEE, and a reverse primer that spans the end of the second exon and the start of the third exon, thus EEEEeeeee), these primers will only amplify from the cDNA, because the sequences eeeeEEEEE and EEEEeeeee do not exist as contiguous segments in the genomic DNA.
Only in the last case is there no possibility of amplifying genomic DNA; this is why you've been asked to design primers in this manner. The first two primer design strategies allow the possibility of genomic DNA amplification, the third design does not.
Only in the last case is there no possibility of amplifying genomic DNA; this is why you've been asked to design primers in this manner. The first two primer design strategies allow the possibility of genomic DNA amplification, the third design does not.
only one of the two primers has to span two exons and the other one can lie in an exon...
-kylvalda-
QUOTE (sonica @ Sep 7 2008, 05:43 PM)
But as iam using exon spanning primers ,syber green will never bind to genomic DNA becoz genomic DNA is not going to amplify to give dsDNA with the primers iam using.Dont you think so?
1. if you have a lot of genomic dna in your cDNA template it will incorporate sybr green
as it will not be amplified by your exon spanning primer pair you will only end up with a more or less high background signal (fluorescence at cycle 1)
not nice but tolerable I guess
2. DNA digestion is no hassle: If you use a kit to prep your RNA it is included anyway, otherwise just do after trizol prep
3. you can check your primers on genomic dna to make sure that they don't amplify there if you want to get around the DNase digestion (to avoid additional pipetting errors, costs etc.)
-kylvalda-
QUOTE (kylvalda @ Sep 7 2008, 01:11 PM)
only one of the two primers has to span two exons and the other one can lie in an exon...
Quite true. But, since it has been mentioned a couple of times that the instructions given are to "design exon spanning primers", I decided to leave the other two equivalent possibilities (only the forward primer or only the reverse primer spans an exon-exon junction) out.
I also agree with your other point, kylvalda -- DNAse digestion is such an easy step, it doesn't make sense to leave it out...
-HomeBrew-
QUOTE (HomeBrew @ Sep 7 2008, 01:17 PM)
QUOTE (kylvalda @ Sep 7 2008, 01:11 PM)
only one of the two primers has to span two exons and the other one can lie in an exon...
Quite true. But, since it has been mentioned a couple of times that the instructions given are to "design exon spanning primers", I decided to leave the other two equivalent possibilities (only the forward primer or only the reverse primer spans an exon-exon junction) out.
I also agree with your other point, kylvalda -- DNAse digestion is such an easy step, it doesn't make sense to leave it out...
If you use a heat-labile DNase, you do not even need to get rid of it.
-Gerd-
Thanks HomeBrew and kylvalda
-sonica-