I have been trying to detect RTLAMP result with HNB dye. However, even though gel electrophoresis confirms my RTLAMP successful, the HNB dye did not change color. The concentration I use is 120uM. I have tried various Mg concentration but it doesn't work, and when I put 4mM MgSO4 the color is sky blue even before reaction so I assume the ph and HNB is right.
The system I use is from NEB:
Tris-HCl PH 8.8 20mM
(NH4)2SO4 10mM
KCl 50mM
MgSO4 8mM
Tween 20 0.10%
HNB 120µM
dNTPs 1.4mM
Primers 1.6 µM FIP/BIP, 0.2 µM F3/B3, 0.4 µM LoopF/B
Bst 0.32U/µl
RT 0.3U/µl
Betaine NA
Enzyme buffer (1.5µl/25µl)
Tris-HCl PH 7.5 10mM
KCl 50mM
DTT 1mM
EDTA 0.1mM
Glycerol 50%
Triton® X-100 0.10%
Thanks a lot for anyone who may provide some input!
Dear Xiao,
Sorry for the delayed reply.
In case, you are still struggling with this issue, I would suggest using a gradient of MgSO4. Like you mentioned, 4 mM, colour is sky blue. But try using 5mM, 6mM and 7mM for the reaction and see if you can get the desired result.
Let us know if this helps or something else that you tried worked for you!
Hello Xiao,
Remember that the reaction buffer also contains 2 mM of MgSO4. In my case (with conventional LAMP), I had optimal color change with a total concentration of 7 to 8 mM. Reactions below 5 mM usually didn't amplify, while with 10 mM or more didn't change color. dNTP concentration also affect the color, as they reduce the available Mg2+ ions in the solution. I hope it helps you. Good luck!
Is it 150uM in 25ul of reaction or is it scaled up to 120mM? Kindly explain
Vivek Krishna P. Venu I guess Mehmet Yüksel refers to using 150uM in 25uL of reaction. The recommended final concentration of HNB is usually 120uM in a 25uL reaction.
Is it possible to use MgCl2 (typical PCR reagent) or MgSO4 should be for LAMP with HNB?
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