Trying to identify the isolate from the high saline area . morphologically isolate is pigmented. extracted the DNA and tried to amplify the 16S. but repeatedly unsuccessful.
1) DNA dilution is done
2) change of primer concentration is done.
3)different primer combination is done.
4)change of PCR cycle is done.
Looking forward to get an answer and successfully amplify the isolate microbe and identify it.
Thanks in Advance
Rehnuma Ghori
Try to perform gram staining. Gram nature may help to select primer pairs. If possible, perform basic biochemical tests like oxidase, catalase, urease, carbohydrate fermentation, identification of pigment. If you are able to classify the isolate in specific Genus, you can be successful in amplifying 16SrRNA gene.
Hi Rehnuma!
As you were previously told, It's probably your isolate belongs to archaea or some kind of yeast. In this case, you have to design or use primers for a suitable rRNA region for them.
Have you verified the integrity of extracted DNA electrophoretically? Sometimes it could be due to lose of integrity in it.
You should also try optimizing annealing temperature, or adding any additive (DMSO, BSA, etc.) to the mixture.
Best wishes!
Hi, Rehnuma,
As the colleagues suggested, it is also important to know what species or group of species you are interested in.
Verify the quality and purity of the DNA using spectrophotometric analysis (A260/280—pure DNA around 1.8) and gel electrophoresis.
If used, the column method for DNA extraction is quite sensitive to samples with extreme pH values (usually higher than 7 pH). Adjust the pH of your samples using HCl or NaOH to ensure they are within the optimal range of your lysis buffer.
To remove residual salts, use desalting columns or additional washing steps during DNA extraction.
Good luck ;-)
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology
- Microorganisms