What is origin of the DNA that you used in your first PCR? Watching your first gel, I should say that your PCR is working well since you do not primer dimers and a good amount of product.

I think that what is not working well is the RT-PCR reaction, and that's why your second PCR do not work well and have many primer dimers. You do not specify what did you run in each well, but in the second line of the second gel you have a specific band of the size you are looking for.

You can try to improve the RT-PCR. Extracting RNA could be problematic sometimes (are you using total RNA, messenger RNA, are you evaporating the ethanol well, are you carring trizol, there are many factors), the purity, the amount of the RNA, the possible genomic DNA contamination, could affect dramatically the outcome of the RT-PCR.

Another posibility is working with the band of the second well, depending of what you are seeking. If you want to clon that band, you can cut and extract it from the gel as it is (I think there is enought DNA for that).

Hope it helps

Hi

please attach your gel picture here ..

Good luck

There are bunch of possibilities that might be causing your problem:

1. You don't have a QA / QC while doing your nucleic acid extarction or PCR master mixing steps .

2. Your primer could have dissociated and rejoined together causing primer dimer .

3. a possible non specific nucleic acid or protein contamination could have happened depending upon the size of the retrieved bands.

4. Following standard QA / QC at all stages ( Extraction stage , PCR stage , Amplification stage and electrophoresis stage ) is an ideal solution for all problems erupting during your PCR work:

a. Check if the water u are using is molecular grade water or not .

b. use sterile Rnase and Dnase free microtiter tips.

c. disinfect the top of your microtiters pipettes with sod hypochrolite 10 %

d. clean your laminar flow or PCR chamber with sod hypochrolite 10

e. Turn on UV lamp on 2 hours at least before your work start.

f. have all of your PCR components (taq, water, minerals , nucelic acid extarct , dntps .....etc ) accurately measured .

g. adjust your amplification protocol (denaturing , annealing , extension , storage temperatures.....)

h. adjusting your electrophoresis solutions , dyes, stains , voltage , time )

Again Quality Assurance / Quality Control (QA/QC) is the key in solving all PCR problems including non specific bands and many others

After gradient you have to check the Amplification again on 58 degree that you got during gradient pcr. different pcr machine having little difference in temperature.

amplify your target product again on 58 and then try again in RT-PCR.

Good luck..

This is the result which I got during my experiment. At first I got band in gradient PCR at different temperature. Please check it. I tried with 58 degree .

What is origin of the DNA that you used in your first PCR? Watching your first gel, I should say that your PCR is working well since you do not primer dimers and a good amount of product.

I think that what is not working well is the RT-PCR reaction, and that's why your second PCR do not work well and have many primer dimers. You do not specify what did you run in each well, but in the second line of the second gel you have a specific band of the size you are looking for.

You can try to improve the RT-PCR. Extracting RNA could be problematic sometimes (are you using total RNA, messenger RNA, are you evaporating the ethanol well, are you carring trizol, there are many factors), the purity, the amount of the RNA, the possible genomic DNA contamination, could affect dramatically the outcome of the RT-PCR.

Another posibility is working with the band of the second well, depending of what you are seeking. If you want to clon that band, you can cut and extract it from the gel as it is (I think there is enought DNA for that).

Hope it helps

Non-specific amplification can be caused by a number of reasons. Your primers look fine based on your gradient PCR gel. Is the template in the first gel genomic DNA? There does appear to be a low level of primer dimer at the bottom of this first gel which you can eliminate by altering the primer concentrations in the final reaction mix. In your RT-PCR gel my first suggestion would have been genomic DNA contamination in your RNA but considering the clearest band is lower than your required band I doubt this is the case. Outside of issues with the quality of your starting template material as described in previous posts you may be amplifying from a transcript with alternative spliced forms. Perhaps try redesigning your primers and see if you can get a more specific RT-PCR reaction? Also you could try running the PCR at a slightly higher annealing temperature.

Hope this helps?

I think you might forget about the core difference between the RNA and DNA in the cell. RNA does not contain introns on which one of your primers could anneal. Thus, you can not get any band. Usual PCR and RT-PCR have different objects of investigations - DNA and RNA which have their own peculiarities and exon/intron structure is one of them.

Looking at your gels, and assuming that by RT-PCR you mean reverse transcriptase PCR rather than real time PCR, and that your marker sets are the same on each gel, then it is clear that in the second gel the topmost band in the sample lanes is a genomic product, in lanes 4 (faint) ,and more prominently 4 & 5. This lies right under a lightening of the gel caused by the use of a UV absorbing maker dye such as xylene cyanol. So it looks like you have some genomic DNA contamination - perhaps quite a lot. Your bottom band is probably a primer dimer or less likely either a shorter splice form than you predicted, or an irrelevant contaminant from off-target amplification of an unrelated cDNA. It appears equally in all lanes , suggesting that the amount of true target available is low, and insufficient to mop up much primer. You do have potential on target products (in your expected size range, but to really understand what they are you need to run more of them on the gel and run the gel longer to get accurate sizing, then purify and sequence.

Before doing all that I would re-optimise the RT-PCR to try to maximise these products - make sure you have no contaminating DNA first by DNase of the RNA prep and then clean up prior to RT-PCR. Then recheck PCR conditions by titrating MgCl2 and by touchdown. Given your first gel result you will probably find you get better specificity at higher temperature: within 2-3C below where you first see a specific signal.

The avoid this problum you should first raised your pcr temprature to at least 40 degree and then keep your pcr tube in the thermal cycler.

Test your primer product by using this site:

http://biocompute.bmi.ac.cn/CZlab/MFEprimer-2.0/

then tyr PCR program from this site:

http://www.mutationdiscovery.com/md/MD.com/screens/optimase/OptimaseInput.html?action=none

Non Specific band are mostly from contamination.

Sometimes primers tend to bind at non specific regions with one or two base pair difference. Check if your target sequence do not have similar sequence .