after nanodrop, I got dna concentration 2324.7ng/ul, then made 1:5 dilution (5ul: 20ul ddH2O); I used 120V (as the distance between electrodes is 25cm), ran 2hrs and half when the loading dye got through half way on my 1% agarose gel.
turns out no visible bands at all except the ladder.
is the concentration a big problem? or because I didn't get any cdna? ( I am using Promega MLV RT point mutant) Any suggestions of cDNA synthesis and gel electrophoresis?
was this a cdna from specific primers or from random primers. If it was from random hex-non amers then your cdna will be of random sizes and spread over a smear in the gel and may not be visible without loading more. If it was in a single band of defined size then you will see your half microgramme comfortably but if spread over a couple of cm of gel it may look very faint
For cDNA it is better to use 1.2 -1.5% agarose gel and run it on 80V (230mV) for 30 minutes.
How many ng did you load on the gel? What is the size of your cDNA?
You should be able to see 50 ng DNA and more. I'd load about 200-300 ng.
To make sure your cDNA size is within the gel, locate similar size bands in your ladder and make sure you see them on your gel. For example, if your cDNA is 700 bp, make sure that 500 bp and 800 bp ladder bands are in the gel.
was this a cdna from specific primers or from random primers. If it was from random hex-non amers then your cdna will be of random sizes and spread over a smear in the gel and may not be visible without loading more. If it was in a single band of defined size then you will see your half microgramme comfortably but if spread over a couple of cm of gel it may look very faint
there is no point doing a larger dilution as it will still not be visible. .If you are happy that all of your material is cDNA then I would use it in your experiments but you do seem to have a lot of material so it would be interesting to see your OD260/280 and 230 ratios just to be sure that the quality of the cDNA is good
The nano drop concentration isn't a true reflection of the concentration. The reason for this, is that it contains a lot of residual primers that contributes to a false concentration reading.
Can I ask why you are running cDNA on a gel? Typically, as Paul has mentioned, using random hexamers gives you a "soup" of cDNA's that look like a smear on a gel. Generally, make your cDNA and proceed to standard PCR to verify that you've successfully made your target cDNA.
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