I have used the same exact sequences in primers while ordering the new primers. The old primers are that I used previously which was worked successfully. Similarly, the sequences in tubes are also the same as that I used previously. Newly ordered sequences are the same and perfectly matched to the sequences that are present in the older primers tubes. The DNA sequences that are present in old primers, newly ordered primers, and even in the sheet have exactly matched. They have no issue. They are also in the same orientation (5’….3’). But, I am not getting any bands using the new primers. I again order the same primers through the different companies but that was also not giving any bands. The other housekeeping primers which are common to both wheat parents, apart from the tested primers have shown clear bands on the same DNA and PCR master mix. The most interesting thing is the ordered primers were not even working right now in the samples DNA that was used successfully in my previous experiment. Then I suspect for the water contamination in my newly ordered primers set that was used to make a 100uM stock solution. I again order new same primers set and prepared a new 100uM stock solution using injection water but it still not giving any bands. I also tried for several ranges of annealing temperatures from 45, 48, 50, 52 55, and 57 whether the annealing temperature has been affecting my results. I am so worried about my experiment, and still not getting any bands.
I used to store all primers, buffer, MgCl2, water, and dNTP mix and Taq solution in the fridger (-20). Even I tried another PCR machine which also didn’t change my result.
Please convey the message if you have any ideas, solutions, and probable reasons that can be affected to this experiment.
I am not a fan of dissolving primers in water even though many companies recommend it. Water allows CO2 to absorb from the air and becomes acidic and depurination can take place particularly if your primer is purine rich although this is usually a slow process. Water can also contain nucleases. Dissolve the primers in TE at 10mM which will keep them alkaline and the edta will inactivate nucleases present.
When you store reagents at -20 it is essential that you thaw them completely and mix thoroughly before pipetting. Salts thaw first leaving floating ice in a partially thawed solution so removing liquid at this stage removes too much salt so the solution becomes weaker every time you may do this. That is a general point and I do not think it is likely to be the reason for your results as other primer sets are working
Hi,
Once I experienced a similar problem. Then I found out Mg is already added with dNTPS. So, Stop the addition of MgCl2 provides me the result.
If you are not getting a band, the primary concern is DNA and Primer. you can check your primer with fresh sample and It would be better if you check the quantification.
Amount of dNTPS and MgCl2 is another issue. Excessive amounts might block PCR.
And finally enzyme. Sometimes we need to adjust our denaturation temp/ time.
and finally, do a longer (more cycles) reaction and see if it gives you bands.
Hope you get your bands. All the best.
I am not a fan of dissolving primers in water even though many companies recommend it. Water allows CO2 to absorb from the air and becomes acidic and depurination can take place particularly if your primer is purine rich although this is usually a slow process. Water can also contain nucleases. Dissolve the primers in TE at 10mM which will keep them alkaline and the edta will inactivate nucleases present.
When you store reagents at -20 it is essential that you thaw them completely and mix thoroughly before pipetting. Salts thaw first leaving floating ice in a partially thawed solution so removing liquid at this stage removes too much salt so the solution becomes weaker every time you may do this. That is a general point and I do not think it is likely to be the reason for your results as other primer sets are working
bonjour. je vous suggère de refaire la PCR sur des échantillons déjà testés positifs mais cette fois en utilisant les nouvelles amorces. si çaane donne rien donc les amorces ne sont plus les bonnes
When I encoutered a similar problem with my primers a few years ago, I discovered that they stopped working due to an exposure of the enzyme I was using at the time to higher temperature than the recommended one due to power failure.
I would suggest testing your primers in a mix containg the DNA samples that have given you bands in the past as well as a new enzyme (either borrowed form a colleague or a newly purchased one) just to be sure.
To me, it does not sound like the issue is with your primers. Throw away ALL of your reagents (water, buffer, Mg, yes the polymerase too), make everything fresh and try again.
The goal is just to get your assay working again, not to solve exactly what went wrong.
I would suggest:
1. Always reconstruct your primers by using Milli Q water or TE. After that make the working solution from this stock again by adding Milli Q water (depending upon the concentration you need). Here is the procedure:
Primer Reconstruction from 100µM Stock Procedure
For the reconstruction of the primers (10µM working) from lyophilized stock (in Fume hood):
(i) Centrifuge the tube containing lyophilized primer at 13,000 RPM for 5 minutes.
(ii) Add __µl* Nuclease-free water to the vial to make 100µM stock.
(iii) Then keep the tube as such for 30 to 60 minutes, for proper primer dissolution.
(iv) Spin for 3 minutes at 8000 RPM.
(v) Vortex briefly
(vi) Take 10µl from this stock in a fresh tube and add 90µl Nuclease-free water ( 10µM primer concentration).
(vii) Spin down/Vortex briefly and use.
*: Quantity of Nuclease-free water to be added is given in the data-sheet of primers in the column ‘µl for 100µM’ corresponding to a particular primer.
2. Always store your chemicals in deep freezer and avoid frequent freez thaws otherwise the primers will degrade.
3. Just before the use in PCR, completely thaw your primers and mix well with the Micropipette and then use.
4. Use some positive control like good quality DNA.
5. Use the previously used primers in some reactions in the same PCR cycle. That will help you in locating the factor that is causing this no amplification issue.
It seems the previous primer is totally different. We often mistaken considering primer sequences from our record similar when the actual sequences on the label of the primer tube are different. So, please be sure that your new primer sequences are exactly similar to that of on the label of the old primer's tube. Then, do PCR using both the old and new primers together. If the old primer still produces bands and the new one doesn't, then the problem is merely with the new primers. If both failed to produce bands, try to change all your reagents. Good luck!
Sir, You should also check dNTPs. I think, it is not the problem of primers if they are same. I have also faced the same problem but as I changed the set of dNTPs , problem get solved.
Dear Rudra Bhattarai
In order to help you, I would ask you to give us all a little bit more info.
1 - Could you send us a good pic of your gel? Using the old primers and the new ones, If possible?
2 - Could you send us your primer sequences (old and new ones)?
3 - Your PCR protocol (reagent setup and thermal cycling)?
With all these in our hands, we can give you better opinion.
Anyway, I do garee with Paul Rutland and Navneeti Chamoli
If you can't share your data, please, review everythingbefore you do new experiments. Check all math on your reagent stocks, even do new ones and take care on dNTP prep ... Once a student working under my guidance did a 4 times more concentrated solution of it and a Multiplex PCR for M. tuberculosis always did get completely negative under this situation. As soon as I reviewed the calculus and found out the wrong amount and correct it, the result, as the paper we got the protocol from presented, appeared perfectly well.
All the best.
- Badges
- Science method