After extracting DNA and running PCR we got very low quantity of DNA. Some of them had no band and some had very unclear band. After measuring on Nano-drop we saw that some samples which has no band had from 0.1-5.1 nanogram DNA in ul, some samples which had very unclear band, hardly but we could see it, they had 2.9-3.0 nanogram DNA in ul. Could you tell me is these quantities enough to be cleaned for PCR? In some cases we even got negative result on nanodrop. What does it mean?
Negative dna usually means thet the nanodop is blanked ( zeroed) wrong. You must zero the machine against the same buffer/solvent that the dna is dissilved in. Elution buffer if it has been column purified.zero the machine frequently in case the electronics are drifting..every 10 samples in large measuring runs and always immesdiately before a measurement run. Clean the reading lens with dilute HCl in case it has got coated with protein over time. If the pcr bands are weak add more dna, run more cycles or design nested primers just inside your original primers and re amplify a first round pcr product ( about 1/10th ul not purified ) with about 20 or less cycles of pcr with the nested primers
Hello George
for standard fluoresecent Sanger sequencing I have also recommended 10ng of DNA per 100bp; Thus, 100ng per kb of amplicon
Thus, I would estimate if your product is small the highest amount could work, but you might experience signal to noise issues, i.e. you will be able to call the bases but it might not be the cleanest sequence in the world
Negative dna usually means thet the nanodop is blanked ( zeroed) wrong. You must zero the machine against the same buffer/solvent that the dna is dissilved in. Elution buffer if it has been column purified.zero the machine frequently in case the electronics are drifting..every 10 samples in large measuring runs and always immesdiately before a measurement run. Clean the reading lens with dilute HCl in case it has got coated with protein over time. If the pcr bands are weak add more dna, run more cycles or design nested primers just inside your original primers and re amplify a first round pcr product ( about 1/10th ul not purified ) with about 20 or less cycles of pcr with the nested primers
You should quantify DNA after extracting DNA and make sure it's concentration between 100 to 150 ng. and do gradient PCR for optimize annealing temperature (for good amount of DNA band).
I concur with Paul. Soak each pedestal for 5 min with 0.5M HCL ( 5ul per pedestal to completely cover ) followed by 5ul of water for 5 min. Between periodic hydrolytic acid washed routinely clean pedestals with DNA or RNA zap or 10% SDS as well as water. Use lint free tissue
Hi George, Try different temperatures for PCR. Also you may want to re-extract DNA from the original samples and redo PCR. It is usually not worth sending for sequencing if there is no band on the gel. The amount you need depends also on the clean up method.
Dear George,
What Paul Rutland said was correct, Please don"t believe nanodrop results. After PCR purification please quantify your products 50ul to 20ul (or less than). Then load 1ul product on 2%gel, If your product will be visible on gel, immediately proceed to sequencing.
try picogreen readings. it is more sensitive than nanodrop. I had similar problem but using picogreen for dna or ribogreen for rna i came to know actual concentration.
Agreeing pretty much to all of above. You need to have good clean PCR product to get good sequencing . How do you clean your PCR products? Are you using any kits or conventional Na-acetate/ethanol?
First optimize the PCR reaction, it makes no sense to try to sequence if no clear band is visible on gel. If it seems to be difficult to obtain a claer PCR product, you can try to sequence with nested primers.
I agree with everyone else. I used Sanger sequencing to sequence many PCR products, and it will only work well if you have one clear, concise PCR band. Otherwise, you will get too much noise to discern your sample's bases. It will be best to optimize the PCR, (i.e. annealing temperature, extension time, DNA concentration, primers, etc), so that you can get the cleanest, brightest band possible. I can also provide you with protocols for my PCR product clean up and sequencing methods if you are interested.
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