i want to amplified Channa striata COI gene using universal fish primer, out of 13 samples only two showed defined product bands after several optimization. I change the PCR mix, change the temperature several times, but i still can't get the desired product bands.The re-amplification of this two samples is working but not for the other samples. The result only showed band with length between 200 bp - 300 bp, even some amplification showed no band at all. i tried to amplified this samples using RAPD and the result is the same. The visualization of the total DNA on agarose gel showed unclear band and smear. I isolated the DNA from muscle tissue using kit.
Does it means that the problem is the DNA template? or something else ? Please if anyone could give me reference of mitochondrial gene amplification of Channa striata or other species from the same family? is there any recommended primers to amplified COI gene from Channa striata or from Channidae family ? Is there any specific primer for Channidae family or channa ? is there any complete sequence of Channa striata gene?
Thank you ,
In my experience with trouble shooting PCR, the reason for failure is usually degraded template or primer problems. The rest of the time it's mostly an inappropriate thermocycling profile. In your case degraded template seems likely. I suggest you test your templates with another set of primers that you know work well.
DNA or RNA extraction from tissues is a little more problematic than from cells since the tissue needs extended homogenisation. During homogenisation DNA and/or RNA can be degraded from the nucleases that are released. Homogenisation in the presence of a chemical that denatures the nucleases e.g guanidinium thiocyanate helps to keep your RNA/DNA intact.
For the specific amplification purpose i sadly can't help you, but maybe for the optimization of the PCR.
Did you try changing the amount of template you use in the reaction? Sometimes too much DNA template can screw up your PCR. Sometimes it also can happen, that your polymerase needs a bit more "support", meaning that it can help to add BSA or DMSO to your reaction mix, but i don't know if you've already tried that.
Also, you mentioned changing the temperatures several times, don't you know the specific annealing temperatures of your primers? Or did you try different temperatures for the polymerase?
Dear Yulia
You mentioned that when assessing the template you found a smear? This usually means that your template is degraded which could explain the inability to amplify consistently. If you have a subset of samples that consistently amplify accordingly it is unlikely that your PCR needs further optimisation and more likely that your template contains impurities or is degraded. The later seems to be the case based on what you've mentioned. You can attempt to purify your product by concentration protocols (phenol/chloroform methodologies) or use adjuvants (betaine, DMSO or formamide) to facilitate amplification but ultimately I would recommend re-extraction.
Good luck!
A useful string to follow for gDNA purification: https://www.researchgate.net/post/Can_I_use_Agencourt_Ampure_XP_for_genomic_DNA_purification
Dear Yulia,
If I were you, I will optimize the annealing temperature for primer. As a thumb rule, set +/- 5 of Tm i.e 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63. Use these temp groups for testing on one sample in 12 PCR column . Run a gel and select the best amplification as Ta (annealing temp) for the rest of the samples.
Good luck
Yulia,
If you are getting a clear band that is different in size from what you expected or multiple bands, it's likely that your primers have an additional target(s) and you may need to redesign your primers.
If you think the problem is with PCR conditions, you can try adding DMSO (up to 5% v/v), betaine (use 5M, add up to 5% v/v) and glycerol (use 50%, add up to 5% v/v). You can different combinations of DMSO/betaine/glycerol, but I generally go for 2.5% or 5% DMSO + 2.5 or 5% betaine combinations first.
You can also try different polymerases. My two favorite ones are Q5 and GXL Star Prime. These seem to work best for difficult templates.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques