pcr product afetr reverse transcriptase reaction - 4 bands i have not included the band which i want??? (Nov/20/2006 )

i isolated rna from infected plant by virus and i made cDNA and i was expecting to have my dna after pcr product, but i didnot have my band and i has 4 other sharp bands.

i dont know why ? if there is any information about that i will be so thankful

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Did you use random primers for RT and specific primers after?

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no, i have used desingned specific reverse primers in RT and then i have used specific forward and reverse primers for PCR

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My experience is quite similar, but I've got other 3 upper bands above the expected (empty) band. I've checked the primers' concentration for the PCR taking into account that you've already introduced the reverse in the RT reaction - presumably not all the primer is used during RT - that depends very much of the volume you are introducing in PCR reaction (mine was 10 in 40 = 50 microliters in total). Also I've done a touchdown PCR to increase the reaction efficiency. Doing that I've "corrected" the situation to 3 bands in total, 1 of interest and 2 more faint bands above the main product. I'm still working to improve the reaction efficiency.

How the NTC looks like? Do you have a positive control? I guess you don't...
Have you performed a Blast search for your sequences? Another idea that I have, maybe the 5' reverse primer is located greater than 2–3 kb from the polyA tail or if secondary structures exist that impede the processivity of the enzyme (it may fail to fully transcribe the template, so that the other primer does not find the aimed complemetary structure to align.

I have no more idea by now

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firstly thank u for ur reply ,
i will repeat this again tomorrow, and i will try to see the difference, what we are doing exactly is to isolate rna of virus which infect plant , from total rna extraction for sequencing and ....,

actually this is the first time for me to work in the research lab and i was afraid that it is contamination. and my supervisor is waiting that i give him good work , to go on with my own way.

i dont have positive control, because we are detecting the presence of virus.
what is NTC?
about the primers , i have taken it from my friend on lab who was working on the same virus and he has its sequence and he designed these primers before.

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NTC means no template control, a reaction in which you don't add any template but ultrapure water instead.
If this completely negative - no contamination, especially with a band as expected mol weigth.
If extra bands still appear - it's mostly like "secondary" products amplify, irrelevant for the target, but reducing the PCR efficiency. You must figure out where these bands come from - more annealing regions in your target sequence, primer dimer, etc. It's quite relevant if the mol wgth is below or above the expected

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