adding glycogen to isoprop for precipitation? - (Aug/10/2006 )
Tried the same and had the same results...
So, as half of the time it's precipitating a restriction I'll indeed add glycogen to water and then use it as a co-precipitant, or just adding it to the restriction reactions itself.
The other half are direct precipitations from viral lysis, so I might add it to the lysis buffer itself, but am not too sure about stability of glycogen in lysis buffers... Any ideas?
Thanx for all thoughts and experiments btw!!!
i just want to say that i learned so much from this little thread! you know, you read plenty of posts , learn a few tidbits that might never come up but this one! wow! multinational science in action- i love it! well done team!
I have premixed linear polyacrylamide with ammonium acetate in order to reduce the number of pipetting steps required for precipitation.
I have question - will the glycogen not effect the DNA , suppose it is used for transfection or restriction analysis?
thanks in advance
Is a of wash of 70% alcohol given to it?
Of course a 70% EtOH wash is part of the procedure. Definately when doing precips with isoprop because you need to get rid of the less volatile Isoprop (and you want to get rid of as much salt as possible).
I have performed Calcium Phosphate transfections of glycogen precipitated DNA on 293T cells and didn't notice a decrease in transfection efficiency. (notice that this was done on a larger batch of cells with several µg's of DNA, so that addition of glycogen would most likely not have improved precipitation too much, I just see the result of this little exp. that glycogen doesn't interfere with this method for transfection on these cells).
About restriction digests: I'm not 100% sure, but I don't think glycogen will interefere too much, but I will compare on monday probably (except if anyone can tell me that it definately does/doesn't interfere).
thanks in advance
According to the Fermentas website...
Features
Optimal for recovery of oligonucleotides (>8 bases) and low amounts of DNA/RNA (>20pg) from diluted solutions.
Free of host DNA/RNA.
Forms clearly visible pellets.
Does not interfere with enzymatic reactions.
Does not interfere with gel electrophoresis of nucleic acids.
Does not interfere with spectrophotometrical determination of DNA/RNA concentration, (A260-280 measurements).
Compatible with DNA transformation and in vitro transfection.
For full details see product description