Hey scientists,
I am intending to observe the extension of a short 10-mer primer on a 16-mer template. I just use a certain DNA polymerase to extend the primer on a templating strand with a matching or mismatching deoxynucleotide triphosphate (dNTP) and the necessary cofactor magnesium.
I was thinking that depending upon the polymerization efficiency, I might have products that are size "n", "n + 1", "n + 2", "n + 3", " n + 4", "n + 5", "n + 6" in size where size "n" is the size of dsDNA duplex without addition of dNTPs, or in other words no extension.
I was envisioning I just combine in a test tube the enzyme with the template:primer construct + the dNTPs of interest + MgCl2 and let the reaction happen for 30 minutes. Then I would denature my protein with heating the test tube to 50°C. (My polymerase isn't one of those thermal stable polymerases), and slow cool the test tube to room temperature.
I could run the products on an ethidium bromide stained gel with a control being a lane with just the template:primer construct with no extension, and then lanes with attempted extension of the dsDNA construct. If at least one base is added I want there to be a notable shift.
What resolution gel could achieve that?
4% Agarose or 16% polyacrylamide or something?
It would be cool, but it's not a big deal for me to distinguish exactly how many bases were extended from "n". So long as I can see an extension from "n" and I would be satisfied.
Thanks!
I would use 20% denaturing PAGE on a long gel ( 40cm old fashioned sequencing gel rig) if possible and silver stain for visualisation. If you have access to a dna sequencer then you could put a long (20-30 base) 5' tail on your oligo to increase its size and dye label the oligo. Then if you run the extension products in the linear acrylamide capillaries it will give you single base discrimination. Size standards are a problem with short sequences which is why I suggest a tailed oligo to bring the product size into the range of the size standards
Pyrosequencing should also be applicable to this problem if your establishment has a pyrosequencer
I would use 20% denaturing PAGE on a long gel ( 40cm old fashioned sequencing gel rig) if possible and silver stain for visualisation. If you have access to a dna sequencer then you could put a long (20-30 base) 5' tail on your oligo to increase its size and dye label the oligo. Then if you run the extension products in the linear acrylamide capillaries it will give you single base discrimination. Size standards are a problem with short sequences which is why I suggest a tailed oligo to bring the product size into the range of the size standards
Pyrosequencing should also be applicable to this problem if your establishment has a pyrosequencer
It's been a while I did this but polyacrylamide is the way, not agarose. The latter will give fuzzier bands while they will be much clearer in the former.
The primers will be easily visible even in the range 5-10% agarose gel but the only problem with such short templates is that the bands are not that much clear so for smaller templates go for PAGE.
Just heat it for 2 minutes max in a microwave then it will gave you ample time to mix EtBr and cast it on a casting plate. Best of luck.
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