It is recommended to store the primers at -20°C. But if someone (forget to keep the primer at -20°C) keep at room temperature for 2 days and then use these.
Q1: Can we use these primer again?
Q2: here is the attached image (File_1.1, compared with the previous results) of using these primers as I could not get the desired results. Could you please tell me is there problem with the Primer or some other issue?
Q3: The faint bands at the bottom are RNA?
I think it is not a big problem to leave the primers for couple days, as I have encountered it sometimes. As for your results, regardless of the short information, I think you used too many matrices, as some bands are seen at the top of your gel, yet it could be simply due to incorrect temperatures or other PCR conditions. It would be better to provide more information and if there are any previous results of the same reaction before and after to compare
As Haidar Fayoud mentioned, not a major issue, however, desired results differ from actual results :) Have you used these primers previously and are the PCR conditions already optimized?
The bands on the bottom are probably primer dimers. If you used a lot of template DNA, the smeared lines could be the fragmented template DNA. The fragmentation could be due to apoptosis or due to processing of your starting material, e.g. cooked food DNA.
Haidar Fayoud Vipul Batra Barbara Brezna Thanks for guidance.
I have edited my question along with the file (with the previous results).
Haidar Fayoud Would you please tell me what do you mean by matrices?
Vipul Batra I habe already optimized the PCR conditions.
Barbara Brezna I agree with you that it could be the primer dimer as it is just 30-50bps band distinct from band of target sequence(which is usually much longer than the 50bps). (i am confused, sometime my professor says that band at bottom is the RNA). Would you please guide me to distinguish bands on a gel and their respective meanings?
Looking forward to hearing from you.
Zeenat Niaz Sorry for the misunderstanding, I meant the Matrix (DNA template), most likely this is an error from autocorrect.
When a large amount of DNA is used up, the polymerase will not work efficiently, as it will find it difficult to reach the DNA strands. The recommended amount of DNA template is 25 to 100 ng per 100-µl reaction volume. To remove all confusion, I suggest that you first measure the DNA amount, then repeat the PCR using the new sample with a previous sample whose result you got earlier and compare them in the same experiment under the same conditions. As far as faint bands are concerned, I think they're primers , it's their typical look. You can add RNAse to remove doubt.
Haidar Fayoud I got you points. Hope so they will work. Thanks for suggestions.
Zeenat Niaz I cannot tell with 100% certainty, whether the bands at the bottom are RNA or primer dimers. I just believe they are more likely to be the primer dimers, based on my experience:
- I sometimes need to clone PCR products into plasmids, and except of correct clones, I sometimes get a portion of smaller plasmids, which have the primer dimers in them, instead of the proper PCR product. (In fact, this one time allowed me to deduce the sequence of commercial copyrighted primers which was kept secret by the producer).
- I sometimes see such bands also in the no template control PCR
- RNA should not be enough in your PCR resulting mixture to create such a strong band. On the other hand, we suspect you used too much template DNA, and if you isolate DNA without RN-ase, it might as well be RNA in your case.
I think it is not important to distinguish RNA vs primer dimers for most people. But if you need to know, look at results of PCR without DNA template (such control PCR should be done in each PCR run) or try RN-ase step in your DNA isolation.
Now I have seen your new photograph.
Which of your line is a PCR without template ?
Barbara Brezna I did not include the control in experiment (PCR without Template). But I understand your point. Now, I will design the experiment with control after checking the quality and quantity of DNA.
Thank you so much