Hi,
I dilute my primers with Milliq water.
My PCR was working fine for 5-6 runs but now it has stopped working. I tried going back to the original stocks in the -20 but the solution has turned yellow. Does anyone know why and is this the reason why my pcr isn't working anymore.
Thanks
If the water was acidic due to CO2 absorption from the air then your primer may have depurinated so becomes useless for pcr. FAM is a fluorescein dye so will be yellow. Primers for pcr are better dissolved in TE to maintain alkaline pH and the EDTA inactivates nucleases. You may have to go back to another aliquot of frozen stock or as it seems that it is the main stock that is not working order a new primer
It is recommended to use TE for dissolving the primer rather than using nuclease free water. Identify the other reason for your pcr, may be problem due to master mix components also. If all going well, sometimes the problems in the lyophilized primer.
Primers are cheap. Throw those ones away and order new ones.
Also, I'd recommend that you make small aliquots of your diluted primers to avoid multiple freeze-thaw cycles. That can also cause primer degradation.
I would recommend storing stock primers in aliqouts in -80deg to prevent freeze thaw cycles and absorbtion of CO2 when you open the tube. Use DEPC water to dilute primers to working solution and try not to dilute too much, 100uL should be sufficient or calculate how much you need for a duration of 6 months.
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