crude DNA was1:10 diluted and used as template for PCR in a 50uL mixture and 4uL of the amplified product was mixed with 1uL of 6x gel loading buffer and run in a 0.8% agarose gel using 1x TE buffer at 80v for 45 minutes. then it was stained using EtBr solution for 15 minutes and destained using 1x TE buffer for half an hour.
maybe, but then the nanodrop readings after pcr were just fine. what might be the reason for PCR failure??when i ran bacterial samples with the same PCR protocol i got amplified product. the dilution was done as i read several papers which told me so. what should be done next to get proper amplified productz?
How large is your product?
try loading about 25µl of your product to check if the PCR really failed. You should always include a positive control (your bacterial samples that worked fine), to make sure your PCR worked.
If there really is a problem with the PCR you could try using more DNA to begin with.
Hello,
in what do ýou dilutet your DNA? Maby in high concentrated EDTA buffer or other wrong buffer? Maby DNA was already degradated? Taken false template tube? Wrong PCR program? And there are many more reasons possible....
Mentioned Nanodrop, maybe you measure only primer, dNTP.
Therefor try again and use a positive and negative control!!!
i think you should use 1.5 % of agarose gel. because maybe amplified product 's size is less. so try with 1.5% agarose gel and dna:loading dye always take 2:1.coc. it will be good for while loading...
see this link for agarose electrophoresis.
http://www.assay-protocol.com/molecular-biology/electrophoresis/agarose-gel-electrophoresis
.8% is fine for small sizes. At 80V it would talk a good 2 hours to run anything off the gel if you are using a normal size gel box. If no bands showed up at all (even those from the ladder) it means your gel did not work.I would put the EtBr in the gel, dont stain with it after the fact. Make your gel (.8g agarose, 100 mL TE buffer, melt it, and once it has stopped steaming, add ~5 microliters of EtBr and pour). If you had bands from your ladder but not from the PCR reaction it means your PCR did not work.
If the ladder shows up in the gel, the process of staining is working. Add a positive control to see if the PCR is working. Make sure you've got DNA by running an agarose gel before the PCR.
In many cases i've found that other products (like humic acids), not eliminated during the DNA extraction, many interfere with the PCR. To overcome this problem, try diluting your samples further, even by 1:100. PCR's are extremely effective procedures that sometimes work with very small concentrations of DNA.
Nanodrop readings may indicate the origin of the problem: lack of DNA, other products, proteins, etc
Check this page
https://www.thermofisher.com/us/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/na-electrophoresis-education/na-electrophoresis-troubleshooting.html
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