I wanted to amplify a 2kb gene with three domains, domain wise. The first domain was around 1 kb, the second was 400bp and the third had 300 bp. After PCR amplification, when I run the gel, I am able to see the bands of template in 1st and 2nd domain PCR product loaded wells, but not even a faint band of the amplified product. The well with the 3rd domain PCR product has shown an intense, good band indicating amplification. What could be the reason for no amplification in 1st and 2nd domains?
PCR conditions were denaturation at 95 C for 2 mins (initial) and 30 sec, annealing at 60 c for 30 sec, extension at 72 c for 1.30 min and 10 min (final). I am using 100ng template.
Hello Sai. Depending on the DNA sequence you wish to copy, the PCR conditions will vary (temperature and time). For instance a CG rich DNA region will need more time for the denaturation step. Also, the primer annealing temperature depends directly on the length and composition of the primer. This means that the parameters have to be adjusted for the different sequences you want to copy. It is possible to calculate the parameters to have an idea (software for DNA analysis, equation to calculate the primer annealing T), then by running a gel, you can see how the result looks, and if it is not what was expected, you can sightly change the parameters and compare the results obtained, until you find the optimal PCR conditions. Good luck.
Hi, how did you extract your DNA? PCR conditions? PCR protocol? Template (how many ng are you using)?...Cheers, Nadine
If you see a template DNA band on agarose (limit ~150-200ng), haven't you simply overloaded the reaction? Maybe the 3rd domain's primers are insensitive to that (comparatively better fit to template)
Hi,
I agree with Marcin that if you use plasmid or PCR product as a template you should lower its amount to c.a. 10ng. Looks that DNA template quality is fine and PCR mixture is ok (you get product for 3'rd "domain").
Some additional tips for PCR optimization for 1'st and 2'nd "domains":
- check for errors in primer sequence and check their proper 5'-->3' orientations,
- too low melting temp of your primers --> try to lower annealing temp (best use a gradient thermocycler for this),
- too high GC content in template encoding 1'st and 2'nd domains - this may cause problems with amplification --> try to add betaine (or DMSO, which I consider worse option) to PCR reaction - this helps in denaturation of interfering secondary DNA structures,
- since you know that your primers for 3'rd domain work try to use forward primers dedicated for 1'st and 2'nd domain in combination with reverse primer for 3'rd one. This should verify if reverse primers for the first two domains are causing problems. (of course extend DNA synthesis time properly),
- try to use other polymerase.
Hope you will find some of those tips helpful,
Lukasz
Hi, You should check the anealing Tm for your primers on website below
https://www.neb.com/tools-and-resources/interactive-tools/tm-calculator
It will give more accurate Tm. You can increase extension time upto too min. the amount of template you can decrease upto 50 or even 20ng. if you have more conc of template, sometimes PCR does not work well due to some unknown reasons.
good luck
Hello Sai. Depending on the DNA sequence you wish to copy, the PCR conditions will vary (temperature and time). For instance a CG rich DNA region will need more time for the denaturation step. Also, the primer annealing temperature depends directly on the length and composition of the primer. This means that the parameters have to be adjusted for the different sequences you want to copy. It is possible to calculate the parameters to have an idea (software for DNA analysis, equation to calculate the primer annealing T), then by running a gel, you can see how the result looks, and if it is not what was expected, you can sightly change the parameters and compare the results obtained, until you find the optimal PCR conditions. Good luck.
Dear, are u confident that your primer is optimized...? if your primer is optimize then run pcr product in fresh buffer.
I'm facing similar problem... !!!
Right now I'm using primer; precisely cloning primer that has leader seq/buffer zone, restriction side and actual primer seq (all are 32bp). It has almost 60oC Tm. After cDNA synthesis and used as template for PCR, then run on 1% agarose no band shown !
Pls what is the solution
I have a problem regarding the PCR. As i run the PCR with different primers but i am not getting the better bands. I revised the process few times but facing the same issue with all of my samples.
can you please help me regarding to sort this issue. As i am doing it in 2% of agarose gel.
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