I have Extracted DNA from six different actinomycetes bacterial strains and then do PCR with 27F & 1492R primers. i should get the band with size between 1400 to 1500 bp. but here i got three bands nearer to each other. now which band should be considered for sequencing to get strain identified. if i send PCR product as it shown on gel, then can i get the informative sequencing results to identified the strain??
here i have attached gel picture of PCR product.
Lane 1, 2, 3, 6, 7 & 8 has PCR product of different 6 samples
where lane 5 has 50 BP DNA marker.
upper most band in marker is of 1400bp, then 1200bp, then 1000, 900, 800 respectively.
you should prepare a new gel and run the PCR product again. The band you need to consider should be compared to the marker for the expected band size. you can increase TIME and reduce the voltage for running the gel. to have a more clear separation
Hi Jibar
Besides repeating the results without changing any parameters, you should change the agarose gel % as well as buffer and voltage.
In addition, you might have to work on the thermo-cycler parameters if you are looking for better results. Make sure you modify one parameter at the time as a reminder and work on the trouble shooting. time consuming but depends how well you expected to be your results. PCR some times are time consuming.
also, gDNA kit extraction could be a problem.
For our 16S we have used 2% gel and it was OK, for the PCR we used 40 cycles of 95deg.-30sec./50deg.-30sec./72deg.-2min. and it was working perfectly for almost all the bacteria. Be sure that your DNA extraction is working correctly with a good quality and quantity (we have used Qiagen) measure your DNA with the nanodrop. With some bacteria we also get some problems. You can also try with a shorter PCR product of ca. 800 bp and amplify the whole 16S in two pieces (primer sequences described in many publications).
I think you should consider gel concentration and running time before doing any other additional confirmation. for the amplification you should consider the more intense band as correct amplified band. you can separate the bands by running a little longer. I normally run at 135V for 25-30 mins and use 1 % Agarose.
Hello Jigar,
I agree with the earlier posts, however there is no point on rerunning these samples on a different gel, it is clear enough to tell that you are getting three bands at the top and the smallest and brightest is the one that is most likely your target as Artur pointed out. Based on the intensity of the bands I am assuming you are planning on doing Sanger sequencing. The easiest option would be to try a few tests to see if you can remove the other bands as Luis suggested. Here are some observations of some changes you can make to your PCR.
1) Increase annealing temperature by increments of 2 degrees C - you are getting plenty of amplification of your target and increasing annealing temperature will increase specificity and may remove that extra bands and still produce enough product.
2) Decrease the amount of primers being used - you have a lot of primer dimer and seem to be amplifying primer artifacts, these artifacts can reduce clarity of the ends of the sequences when sequencing.
3) Reduce the amount of template DNA - you have a smear of DNA in the background indicating that you may be using to much template, reducing the amount of template can sometimes also decrease the chance of amplifying these secondly bands and increase specificity.
4) Reduce number of cycles - you are getting plenty of amplification of your target so reducing the number of cycles may reduce the secondary bands while still getting enough of your target.
I would probably start by reducing the primers by 10-25% and do a temperature smear. If you have a gradient thermocycler I would set it for increments of 2C starting with your current temperature and then 2C, 4C, and 6C higher annealing temperatures in the other lanes. If not I would just start running tests of 2C and 4C higher annealing temperatures and then try some of the other suggestions if this does not work.
Hope this helps.
Best Regards.
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