I am looking forward to receiving your comments and ideas and suggestions
Yes, that "heavy product" is the template DNA and is obviously too concentrated, otherwise you won't be able to see'em such clearly. I agree with Paul: "your PCR is not working". Try to follow the suggestions stated above and test the DNA concentration before preparing PCR reactions. Put no more than 100 ng DNA per 25 ul reaction.
Your PCR product is too high. Maybe your used primers did not find complementary site. Or your PCR didn't work well. You should optimize it or change your primers in order to amplify properly your selected fragment.
the large size bands are either poorly purified dna possibly with protein still attached. Your pcr is not working. I think that the small bands are a mixture of primer dimer and rna. Run a no sample pcr should show if you have primer dimer. If you let us know the primer sequences and your pcr conditions you will get plenty of advice. it is likely that as you have rna present you are not usingg enough dna because most of your sample is rna and possibly you need more cycles but mostly you need to examine your pcr parameters
Hi Khoo,
What is the size of your template DNA? How much template you used? The upper band could be your template. In this case you need to optimize you conditions to get the specific PCR product (300 bp. If the upper band is a PCR product, you need also to optimize your PCR conditions or redesign new specific primers. Wish you good luck.
The upper bands are very similar to DNA . As Paul said, the lower bands seems to be a mix of primers and RNA . I think you used too much DNA in your reactions. Do you agree with me Pegah ? :)
Yes, that "heavy product" is the template DNA and is obviously too concentrated, otherwise you won't be able to see'em such clearly. I agree with Paul: "your PCR is not working". Try to follow the suggestions stated above and test the DNA concentration before preparing PCR reactions. Put no more than 100 ng DNA per 25 ul reaction.
I do agree with the rest regarding the smaller bands, degraded RNA and primer dimers.
Now the upper band...
you definitely need optimisation here.
1) DNA. Are you using cDNA or genomic DNA?
2) DNA concentration. Depending on the abundance of your gene you should use around 5-20ng of starting material per 25µl rxn.
3) Which region of the gene are your primers designed? are your primers exonic or intronic? Usually, when possible, primers should be designed to span an intron to help exclude any issues with genomic DNA contamination.
4) if you are doing RT, do you know it definitely worked?
5) do you have a positive control for the gene of interest?
6) primer optimisation regarding Tm and Mg2Cl? these could help with specific amplification
7) are your reagents working ok?
good luck
I have a question too.
I am working by ISSR markers and my ladder is 100bp.in PCR gel some band are above the ladder.is there any problem for scoring them when I don't know their size?
Should I use another ladder(for example 200bp)?
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