30μl reaction components include, DNA 2μl, Master 15μl, Primer 0.7+0.7μl, water 11.6μl
PCR program
98°C 2min 1
98°C 15S 35
56-61°C 20S 35
72°C 30S 35
72°C 5min 1
How much dna in nanogrammes are you using. Sometimes too much dna can be inhibitory to pcr. It is worth running different dilutions of dna.Somewhere between 25 and 50ng might produce good amplifications
try a pcr with 12, 25 and 50ng dna. the dna may have pcr inhibitors present so less dna means less inhibitor so mor pcr product
You could also try adding more Mg ions.Many pcr inhoibitors take MG out of the reaction and make the enzyme less active.Try 0.5mM extra Mg.
Betaine and dmso are very useful in reducing secondary structure in AT rich or GC rich sequences.I normaly used a final concentartion of 1M betaine and up to 10% dmso....6% is a good place to start or run samples with 1%, 2%......8% dmso. If you do not have these reagents to hand then most high GC additives that come with polymerases are a mixture of betaine,dmso and sometimes BSA and DTT so any high GC additive will help if your dna has a lot of secondary structure
yes try adding an extra 0.5mM of Mgcl2. This slightly lowers the annealing temperature of the primers and also counteracts any effect from pcr inhibitors in your dna. Some modern long amplimer polymerases are a bit fussy about Mg concentration but extra Mg works ok for most polymerases
Hi Reza Kh ,
if you're sharing your PCR protocol, you should include also stock concentrations or final concentrations of your reagents, because volume is useless information (are your primers 100 uM or 1 uM?).
Similarly, to judge your PCR cycling, we need to know what is the Tm of your primers and length of your amplicon. If you keep having troubles after implementing Paul's suggestions, you may try to decrease annealing temperature or do touchdown PCR.
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