basic basic question regarding pcr product storage - (Oct/09/2008 )
a long time reader, first time posting
needing help with really really basic stuff - that I'm almost embarassed to ask
(I should probably be asking this to my sup, but we just don't get along)
ehm... for pcr amplification products... I'm amplifying the insert for sequencing, and use just about 0.5 ul out of the 20 for the total rxn...
does the amplified insert decompose at roomT?
**I'm really interested in getting the whole picture... like the covalent backbone and the hydrogen bonds btwn base pairs interactions and how they are strong enough (or not) to keep from decomposing... blah blah blah
also, for the sequencing PCR... the products, why exactly is it we want to prevent their exposure to light? is it about degradation because of the single-stranded nature of the product, or does it have to do with the ddntps fluorescence?
thanks a bunch you guys,
Your DNA will not decompose at RT (if you don't have nuclease contaminations). You can keep the DNA for days at RT.
You probably want to protect it from light (sunlight) because UV can cause mutations and that would give you a wrong sequencing result.
I would recommend keeping the PCR product in -20 degree . it won't hurt and DNA is more stable that way ( they don't move around much frozen, " static" condition = good) and if u need em , just need to thaw for like 10 sec.
Light exposure is bad for the fluorescent tag in the Big DYe. not really related to DNA but then again UV is also bad for DNA.