Two bands with MSP - (Feb/05/2006 )
My name is Kinga , I have ben working on MSP for 2 months, I designed my primers to the promotor of gene of interest and first I obtained perfect result -amplified product only with primers designed for unmethylated template, but I wanted to improve it so I went higher with magnezium chloride and the number of cycles in order to have the stronger band and after all I got two bands, Do you think it is the result magnezium and the higher numberof cycles?????? because I find it dificult to explain
thanx for your answer
The second band would be due to the magnesium.
If you want to improve your original PCR (the one which worked fine), try adding a few more cycles.
Increasing the magnesium can result in amplifying non-specific targets that your primers have some similarity with.
tanke you Davo I came back to the previous concentration of magnezium I will see the results in around two hours I will tell you what was it
now I have one band again but very very weak could you tell me how to increse the signal without loosing specificity????
I will be grateful
apart of adding the cycles this was don ebut still the signal is not suficient
Maybe you can try to run two round PCR by using your amplicons from your 1st PCR as template. Since you have very weak band, try to put 2 to 3 micolitre of your 1st amplicons. Hope this may help
[thank you pcrman I will try to do it