I am testing ISSR primers on plant DNA and the bands I get are very faint. I am adding 5 microliters to the wells from a 25 microliter PCR product, I have tried increasing the volume in the well but I get same results. Does anyone have an idea what could be causing very faint bands?
Hi! looks that you have low DNA concentration in PCR product. Try to load less amount of DNA ladder, it can be an issue with some geldocs.
Hi Hadeel,
Try different DNA concentration by calculating how many ng/miro-L DNA you have in a PCR reaction.
Dear @ Hadeel Ossama Mohamed, i did not use ISSR, i did SSR markers, but i think your problem is about your PCR reaction, as you see, your bands are very thin and near together, with low distance ok?, so i think maybe if you up or high your annealing temprature, please attention maybe if you low this temp maybe you see result, because when i did it, i understand some my SSR primers are better result in low temprature of annealing, if no problem please say family of your plant, if it is near Malus (Apple), low temprature is better, maybe 47 or 50 was better. Please remember i just said just my probabilities maybe are related to your bands in picture. I hope you find better result.
Ah i forgot, when i reduced concentration of MgCl2 and dntp, and add amount of primer, i had better bands, see please:
At first i used this :
1 micro liter MgCl2
0.5 // dntp
0.5 // Forward and reverse
But when i changet it to following, i saw better bands really:
0.3 // MgCl2
0.1 // dntp
0.8 // Forward and reverse
If your DNA had good quality, i think better you focus on stuff effect on annealing primer to your DNAs...
DNA concentration is low in your pcr production, you can try to increase your pcr cycle number, or add more DNA template.
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