Is my plasmid contaminated by RNA? - (Dec/02/2006 )
hello mates,
desperate help needed due to a horrible tutor 
. we isolated plasmids using boiling and alkaline lysis methods i cant figure everything out in the photo/ i guess i got samples contaminated with RNA at 1,4,5,7
any help will b great,
cheers,
have a nice weekend 
You have RNA and genomic DNA contamination in your samples.
Don't vortex after addition od solution2 (NaOH+SDS) and use RNase
remove supernatant
vortex the cell pelllet first. (this breaks up the cell pellet)
then add solution I + RNAse
then vortex for a short time. this is to mix the cells into solution
then add solution II
to mix the two, just invert the tube 5-10 times
then add solution III
you can vortex lightly or invert tube to mix
Don't vortex after addition od solution2 (NaOH+SDS) and use RNase
I followed instructions. as i said i have a really bad instructor. can i still figure out the length of the plasmids? which one is what?
You have RNA and genomic DNA contamination in your samples.
Don't vortex after addition od solution2 (NaOH+SDS) and use RNase
I followed instructions. as i said i have a really bad instructor. can i still figure out the length of the plasmids? which one is what?
The lowest fat band is RNA; the material stuck into the well and creating the smear along the lane is genomic DNA. In order to determine the size of your plasmids, you need to linearize them by cutting with a single cutter. With such a contamination, you can use these plasmids neither for transfection nor cloning.
is it a mini prep or large prep by alkaline lysis method? if it is a mini prep and the DNA sample is just to use to check some ligated plasmid then contamination of RNA is not a very big problem.
but if it is a large prep sample then u should perform additional RNase treatment
Hi,
It's going to be messy to do RE digest if you have gDNA contamination, but you could try anyway. If you have supercoiled DNA ladder, you can estimate the size of your plasmid by looking at the lowest band (supercoiled) of your isolated plasmid too with RE digest (fast estimation).
It's going to be messy to do RE digest if you have gDNA contamination, but you could try anyway. If you have supercoiled DNA ladder, you can estimate the size of your plasmid by looking at the lowest band (supercoiled) of your isolated plasmid too with RE digest (fast estimation).
what about whats in the sink, where the samples are loaded? can i assume it's dna fractions?
Hi,
It's going to be messy to do RE digest if you have gDNA contamination, but you could try anyway. If you have supercoiled DNA ladder, you can estimate the size of your plasmid by looking at the lowest band (supercoiled) of your isolated plasmid too with RE digest (fast estimation).
what about whats in the sink, where the samples are loaded? can i assume it's dna fractions?
The sink where the samples are loaded is the well. As I told you, that's genomic DNA
Hi,
Typo error: it's "without RE digest when using supercoiled DNA ladder for fast estimation".
DNA near the sink (or well), as dnafactory described, is gDNA. It's too large, therefore migrated slowly.
Do you mean plasmid conformations, e.g. supercoiled, nicked and linearized? Nope. It's too high to be linearized plasmid.