cDNA precipitate & DNA ladders - (Mar/30/2006 )
Silly beginner question about PCR: why is cDNA precipitated? If it is for storaging, can it be stored in soln at -20C or -80C?
Also, when trying to decide appropriate DNA ladders, where do you find info regarding the size (bp) of your product? I realize that sometimes they are in the literature, but it is quite variable whether or not a gel is included in the article, etc. Is there a quick resource you guys/gals check for this info (length/size of product in 'kb')?
Most people store their cDNA at -20 in solution, -80 is fine too (avoid freeze/thaw as much as possible) I have never precipitated it unless it needs further cleaning after the prep.
The size of your product will depend on the primers you are using, it will be the length between the start and finish of the primers on the cDNA sequence. If you know what your target cDNA is (and you should), then you should be able to find the sequence on Genbank
here and align your primers using an alignment program such as Vector NTI (see invitrogen) or any other for that matter, that will give the size of your product.
Does the precipitate soln itself 'clean up' the DNA, or is that only done by something like one of the Qiagen kits?
Thank you for the GenBank link; much appreciated.
no, the precipitate soln (if you mean EtOH and NH4Ac) doesn't clean up the DNA. You can wash off the salt by washing the pellet with 75% EtOH. You can do phenol extraction before precipitation to remove proteins.
Qiagen kits usually utilize the different binding properties of DNA/RNA/ protein with the resin under different pH values and salt concentrations to clean up the DNA/RNA.
That's what I figured.