I am trying to do LAMP reaction using phenol red, however, even using low buffer concentration or no buffer I can not see any difference between positive and negative controls. After LAMP amplification my master mix keeps the same pH. Does any one can give me some advices?
Dear Juliana Cristina Borba,
Please check this attached file.
Best regards
did you run any agarose gel to see if amplification occurs in the positive control?
Eleni Makraki Yes, We did! The positive control worked.
Mohamed-Mourad Lafifi Thank you!
Juliane Cristina Borba make sure then that you don’t have buffer carry over of the enzyme’s storage buffer (you should use high concentrated enzyme stock) /primers/target in your LAMP reactions. You can also double check that you phenol red solution is ok
Thank you Eleni Makraki !! Actually we realised we were having buffer carry over from the RT to obtain cDNA!! We were able to remove that and the phenol red worked fine!! Thank you for your help!!
hey! maybe you can try with others dyes like HNB, because is less sensitive to changes in condition (pH detection dyes like phenol red doesn't work with some extraction condition, specially NaCl based methods).
I'll attach a photo of color changes with HNB dye, its such a beautifull
dear Eleni Makraki I have the same problem and I work with warmstart master mix 2 x 1804. I think the problem is related to poor reaction amplification that could not change the pH of master mix. I am eagerly waiting for hearing your answer.
Razieh Taghizadeh can you please share the protocol to prepare phenol red /HNB and the LAMP reaction using New England BioLabs E1700? We want to try a colorimetric read out with tha same kit.
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