I have a very critical problem related to my above mentioned work. Outer primer specific to this pathogen having amplicon size of 1.8 kb and two pair of inner primer have 1.2 as well 0.8 kb. But when I go for nested PCR I get multiple banding pattern even after nested PCR in some tissue of plant having infection with this particular pathogen, As well as also get specific band in some tissue after nested PCR in same plant host. So why we did not get the same at all tissue of plant?
I am unable to troubleshoot this problem. Can anyone help me for troubleshooting this problem?
Nested PCR means you will need two sets of primers to do the overall PCR DNA reaction. You will do one reaction at first, and once you get that product, you will apply the second set of primers. The idea if to get a more pure end product of what you are looking for it. There a few disadvantages of nested PCR: cost and time. For example, when you do it manually it takes a lot of time, so it will be best when you will have many samples and the use of automated instrument. But again, the nested PCR instruments can be costly. Another disadvantage is that sometimes the end product could be contaminated. You will need really to set good rooms to run nested PCR
Did you dilute your PCR product before you performed your nested PCR? Diluting (e.g.1:100) your PCR product will make sure the excess primers from your first PCR reaction will not interfere with the primers from your nested PCR reaction. Hope this helps.
Thank a lot to both Karin G Hermans & Yvette Ghannam for there precious suggetion. I already performed second PCR with dilution as recommended you, even I didn't got reproducibility in results.
I agree with the other comments. 1:10 to 1:100 dilution of the first PCR is advisable. You should also proceed carefully to avoid cross contamination in further experiments when working with nested PCR. Separated working areas should be set for each step.
I can refere you to study one of my articles which included Nested_PCR Method. I hope it can help you effectively.
Are you sure your primers are specific for your pathogen of interest? You can do a blast search to see if your primers bind to anything else.
Thanx Seyed Shervin Shariatpanah& Karin G Hermans................ yes second primer is specific to pathogen but outer primer some time bind with homologous pathogen also
Seyed Shervin Shariatpanahi sir, please tell me about your related articale
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