I am trying to clone a T.thermophilus gene. I have used 3%/5% DMSO during PCR amplification (using phusion enzyme). Amplification is coming but after PCR purification, and I am also getting non-specific bands. Suggestions are needed to avoid these non-specific bands and primer dimers.
Why dont you cut that band from the gel purify it and clone it. this is best strategy or else you can try by increasing the temp for primer annealing. standardize annealing temp by different gradient temp in pcr.
Why dont you cut that band from the gel purify it and clone it. this is best strategy or else you can try by increasing the temp for primer annealing. standardize annealing temp by different gradient temp in pcr.
As said above try to increase stringincy of reaction... And go for gel purification...
Pankaj makes sensible suggestions. You could also replace DMSO with BSA (1 ug/ul). This is an alternative way of reducing non-specificity in your PCR reactions.
check your primer sequence using the primer-blast server. note that even sequences with 3-4 mismatch bases would possibly be amplified in the reaction,
PCR cleanup kit will remove any unwanted salts and proteins, not those unspecific bands. i would suggest ignoring the non-specific band and extracting the correct size from the gel using a gel purification kit.
good luck!
Given my experience with Phusion Pol, I've been getting cleaner results and better efficiency by using 0.5-1M betaine (Sigma_Aldrich) rather than DMSO.
Dear Suraj
Well prc cleaning method is other option to get your desired product. But i will advice you to take of care of template DNA, optimize primer concentration. annealing temperature and time for primer.If you will go through, these parameters you will hardly get any problem in PCR amplification in future.
@andrei and @Pankaj: thanks to you and other people also for their suggestions.
I have already tried what pankaj has suggested but after gel extraction the concentration decreases to a greater extent so as I cannot proceed with the digestion and further steps.
@andrei sir: According to your 2nd situation, if these are the artefacts then should I continue with the digestion with that sample only or I have to have a single band (only gene of interest)??
You can directly try for PCR product cleaning kits, read their manual carefully, many times they specify volumes and different incubation time with binding buffer with respect to your band size.
Just try it.
Good Luck!
ideally or logically, after gel extraction of your specific amplicon, you should check it on gel in such case you will see a single band. if u see nonspecific bands, that means during the PCR/Amplification reaction, many similar sized products are generated. After gel extraction you can go for RE-PCR or when you see your target band after gel extraction, elute it again and use it as template for Re-PCR.
doing this may help you to recover your specific amplification.
regarding kits for gel extraction, you should use Qiagen gel extraction kit. its really good and of best quality approved worldwide across well established research groups.
Suraj,
In case you have a single PCR product band, I suggest sequencing it to verify the real content of the gel band. As Andrei says, a single band may contain several products. A really specific single band should provide a clear good quality sequence when PCR primers are used in the sequencing reaction. PCR primers very careful design and an extreme care in the anti-contamination PCR procedures may be necessary.
If your PCR product show more than one band, you must cut from agarose gel the interested band having the correct size and purify it using a specific kit.
To avoid non-specific PCR product, you can re-optimize your PCR conditions especially the T°C of annealing (use Tm calculator). You can also do nested-PCR.
Good luck.
If you can't get a clean band through gel purification, I would suggest that you just clone your PCR product, do a PCR of the clones, and only sequence the clones that are fragments of the right size. Once you have a clean sequence, you may be able to optimize your primer sequence avoid non-specific product in the future.
If you are having trouble with excising the fragment of the correct size, I would recommend a product from life technologies called e-gels size select.
Two options:
1. Optimize your conditions to remove non-specific bands (lower template or polymerase quantity work well)
2. gel purify samples (like was suggested above: if it is only a question of poor yeild, you can brute force the approach to get lots of gel purified product). Or perform a PCR on gel purified samples at a higher annealing temperature.
Some other options you could try are a different polymerase (Pfx, Vent), or altering the magnesium concentration in the buffer. If you want to have more PCR product to work with, set up multiple small (15-20 ul) reactions, then pool them for the cleanup steps. I've had more success with this method than with setting up larger volume PCR reaction. Good luck!
I agree with Steve, you have to try minimize the template volume, and revise your PCR conditions .
Good luck!
hey got the clone.. thanx to everyone for their help...
special thanks to pankaj and steve and to everyone....
I imagine your DNA is very GC rich if it comes from T.thermophilus. You will need a very high annealing temperature to avoid non-specific bands and even this may not help. Just cut the appropriate band out of a gel as others have suggested.
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