since i am working with ssr markers I am not getting any kind of amplification though conducted series of veriflex, gradient pcr. Please help me out with this
You need to read the instructions for the mastermix but most come at 2x concentration so use 10ul in a 20ul reaction. If your pcr is not working it is likely that either the primers or the dna is poor quality so you need to sort out which. Borrow a dna from a colleague that amplifies with any other primer set and use this to test your primer integrity. You could try dna at 5-10ng and primer at 0.1 to 0.5 uM ( that is 2pmol to10pmol of primer per reaction. Water makes up the remaining volume
Subash Thapa Ask for help of technician or seniors in your lab. The best and easy fix to your problems.
Danial Shokouhi If you refer to Paul Rutland 's answer, please read it and understand it carefully, before adding information of your own. Did you yourself see how much DNA you are advising to be used in the reaction, and what would be the final DNA concentration in PCR reaction in the DNA stock has concentration of 50-100 ng/µl? You are advising to add too much of DNA which would anyway inhibit the reaction.
If you are using the master mix for a 20 µl PCR reaction.
You should add
1. 10µl master mix.
2. primer 0.05µl each forward and reverse. (first, dilute the primers10 times from the stock provided by the company).
3. template DNA 1µl (If isolated by using DNA isolation kit otherwise 0.05µl if isolated manually)
4. 8 µl nuclease-free water.
This protocol I have used and standardized.
Subash Thapa Bilal Ahmed
Mohammad Tarique Zeyad
Seriously?
I haven't seen this sort of detailed instructions about DNA template without knowing anything about the samples. The point 3, it is just absurd.
Since i am working with fungus DNA samples and have done quantification by running them in 0.8 % agarose gel. Though good bands were formed and i lack nanodrop to go for quantification. What should be the dilution of dna for working solution?
Subash Thapa
As I suggested before, Ask for help from technician or seniors in your lab. The best and easy fix to your problems.
Further, what do you want to do with the DNA?
If you don't have nanodrop in your lab, ask for analysis in some other lab. I don't think there is no Nanodrop in whole BHU.
@Abhijeet Singh
I am working on molecular variability among the fungal isolates. Though nano drops are available but labs are closed due to covid.
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