My problem is related with ITS primer .Previously i had no problem but from fews days bands on 1% agarose gel sometimes show smears, sometimes multiple bands and sometimes increase in amplicon size. I checked all the reagents and conitions ,and found when used i use buffer and mgcl2 from others lab (and other reagents own) , i got no problem .But when i use either buffer or mgcl2 of own i got above problems
I would think you have contaminated supplies with either DNA or other oligos in it. I would suggest to start with a fresh batch of everything if you can (oligos, buffers, taq, water, dNTPs). In the past I have split up the original solutions into smaller aliquots so when I noticed a contamination I could simply throw it away and start with fresh supplies.
Thanks Muzafar , but there is no issue with gel percentage as 1% gel is compatible for ITSwith amplicon size of 750
Thanks Natascha Mam . Actually my query is does problem in either buffer or MgCl2 or in both causes these uneven results
there are 2 main possibilities.
1
one of these reagents is contaminated with previously amplified pcr product so you are re amplifying this and there is now so much product that the product is self annealing and producing smears of long size amplimers.
2 the other possibility is that you are not letting your magnesium thaw completely, The salt melts first so if there is ice left in the tube or if you do not mix the magnesium before pipetting then when you pipette out a volume of solution it is mainly water/buffer from the top of the tube or Mg from the bottom of the tube. In this case taking water from the top makes the Mg concentration higher so the primers will anneal at a lower temperature and many wrong bands will be amplified as the \mg concentratio gets higher in your tube
I think contamination is the mre likely. Does your zero sample blank produce smears or bands?
Should check the buffer you use in electrophoresis and voltage. If is to high may denaturation of your amplicon.
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