(LAMP) Problems with Loop-mediated isothermal amplification

[Edok]

Hello all,
thanks a lot for your posts in the discussion about LAMP. I have started with the assay but my problems are as the problems I saw you discuss. First is a series of false positive amplifications even as much as I try them. I am also not able to generate any bands when I use primers with wobble bases. I saw you talked about melting curves using sybr.green, how do I do this?

As much as I can generate sometimes amplification which is observed in the under UV and also in the gel, I am not able to see any visual turbidity. Could you be of help (Apart from my primers, the rest I use an RNA extraction kit)

Thanks in advance

[mcalapai]

So I actually don't use the melting curve. So I'm not sure what that would be.

As for me, It worked yesterday with all new reagents and primers with HNB. Not sure what changed but I am going to try to repeat this.

[mcalapai]

EdOk- I wasn't able to observe turbidity either. I had a lot of problems then, What are the concentrations of your reagents? Maybe it is a problem with primer concentration, MgSO4 or betaine, or all three? Maybe try switching it up?

Hope thats was helpful

[msarah]

I used both sybr and Eva green. Worked the same. I have only one ( or two depending on if there is false pos or not) MT peak.

[Edok]

Hello Mcalapai & Msarah, thanks a lot and sorry, i was not able to respond on time. I am using the extraction kit from a company and another one from Eiken Japan. I am not sure about the concentrations of the other reagents. I am trying to use primer conc of F1 and B1 0.2M each, FIP and BIP 1.6µM each and 0.8µM of the loop primers. I cannot see any turbidity.

Probably are u able to give me an idea on how to make a primer mix for all the six primers and they still retain their concentrations in a 25µL reaction? Maybe the amount I use (totalling 4 µL for all the primers) could be part of the problem.

[msarah]

Reagent Stock Conc Final Conc µl/25 µl
Sol. 1
Tris buffer pH8.8 1 M 20 mM 0.5
KCl 1 M 10 mM 0.25
MgSO4 500 mM 8 mM 0.4
NH4(SO4) 1 M 10 mM 0.25
Tween 20 10 % 0.1% 0.25
Betaine 5M 0.8 M 4.0
Manganese(II) chloride 50 mM 0.5 mM 0.5
dNTP mix 100 mM 1.4 mM each 1.4



Reagent Stock Conc Final Conc µl/12.5 µl
Sol. 2
FIP 100 µM 40 pmole 0.4 µl
BIP 100 µM 40 pmole 0.4 µl
F3 10 µM 5 pmole 0.5 µl
B3 10 µM 5 pmole 0.5 µl
Loop B 10 µM 20 pmole 2.0
Loop F 10 µM 20 pmole 2.0
Bst DNA pol 8 U 1.0 µl
DNA 1.0 µl
H2O 4.0
25 µl total

[Edok]

Hello Msarah, thanks a lot for this post. For sure I will try it as well. For sol. 2 I used the following and it seemed to work as well;
FIP 80µM 40 pmole 0.5µl
BIP 80µM 40 pmole 0.5µl
F3 20µM 5 pmole 0.25µl
B3 20µM 5 pmole 0.25µl
LF 80µM 20 pmole 0.25µl
LB 80µM 20 pmole 0.25µl
Dye 1µl
Bst polym 1µl
Reverse Trans 0.5µl
RNA 3µl
water 5µl

It seemed to work. However My main problem is how to make calculations and make a working solution of the primer mix where I am only able to pipette like 2µl or 2.5µl for the reaction and maintain the concentrations of all the primers. In what quantities can I combine these different concentrations so as to have a mix that I can only pipette a standard quantity for the mastermix? Once more have you been able to observe visual turbidity without the use of any dye? Thanks again.

[msarah]

To see sediment you must add Manganese(II) chloride to the reaction. We have the best results with HNB, but only for strong reactions. In the weak reactions you need to read OD.
Concerning primers, we make a large mix of all three primers together and freeze in aliquots. This way you are adding all the primers together and the volume is larger. You can always order your primers freeze dried (Sigma) and dilute work stock to whatever concentration is easy for you to work from.

[deen23]

Hello,

I'm currently trying to find the optimal concentrations for an RT-LAMP assay. I've tried many different combinations, but now I'm doing a total mixture of 25uL with the following concentrations:
FIP 1.6uM
BIP 1.6uM
F 0.2uM
B 0.2uM
dNTPs at 1.0mM (I used to do 1.4mM)
Thermopol buffer 1X (which contains 2mM MgSO4)
Betaine 0.8M
MgSO4 6mM (to make a final total of 8mM in solution)
Fluorescent detection reagent 1uL (from the Eiken kit, no concentration specified)
Bst DNA Polymerase at 8U for the rxn, or 0.32U/uL
3.5uL of RNA sample (I've been using from 1uL to 5uL)

My problem lies with the amount of reverse transcriptase to use. Papers state very different amounts, and the RT-LAMP kit that I was basing it on doesn't specify its concentration. I'm using a Cloned AMV Reverse Transcriptase from Invitrogen (products.invitrogen.com/ivgn/product/12328019) which has a concentration of 15U/uL, and comes with 4 other tubes/reagents, one of which is DTT. I was told that I must use DTT at a final concentration of 10mM. However, I don't know how many units of RT I need in my reaction. Could someone please let me know what they are using?

Thank you so much!

[Babak]

I was working with HNB for my LAMP solution now. Before using HNB, my LAMP samples works properly and it amplifies well but once I add HNB to my solution , none of my samples works and it seems HNB inhibits the amplification. I was wondering if anyone faced with this problem? Could someone help me out with this issue?


Thanks
Babak