(LAMP) Problems with Loop-mediated isothermal amplification

[nikan]

Originally Posted by butters

Molecular grade chemicals have not nuclease or rnase activity detected. Other chemical that have these activities may break you DNA and causes some problem.

By all mean you want to go ahead with it since you have prepare the solution. If you have the MgSO4 that come together with your Taq, then you can do a comparison. Maybe it will work for you.

Thanks. You are certainly right, molecular grade chemicals are RNase or DNase
free. I though if I autoclave it, DNase would be destroyed. I am not sure whether by autoclaving we can remove RNase or not but I think DNase can be removed. So we wont have DNase to break down DNA.
Do you know if I autoclave it several times it will cause any problem or makes it better?

[nikan]

You are certainly right molecular grade chemicals are nuclease free but you know I thoght autoclave would remove DNase not sure about RNase but I think DNase will be destroyed. what will happen if I autoclave it several times?!

[nikan]

Dear friends,
I have a question and I would appreciate it if you could help. How should I dilute SYBR green for LAMP? Its concentration is 10,000x and if I am going to use 1ul of it for each reaction how should I dilute it? Some body told me 1 ul of this SYBR with 10 ul water will give you the appropriate dilution or 1/10. Is it correct?

Thanks in advance

[butters]

Nikan you can use M1V1=M2V2 equation for the sybr dilution. M=concentration, V=volume, 1 stock volume/conc, 2 target volume/conc.

[nikan]

I used the MgSO 4 and the turbidity was ok. I think nothing was wrong with it. I had two turbid positive controls, the negative control seemed not to be turbid. But when I electrophoresed LAMP products my negative control showed a slight ladder pattern. The pattern is different from my positive control. It seems that there have been contamination. Do have any idea to help. The problem is not with my primers since I took if from an article.
Thank you

[nikan]

Can it be beacuse of not heating for 2 min at 80 degree after the termination of reaction????? I skiped this step and electrophpresed it after 10 min keeping at -20 degree.
Hey some body help!

[Cicici]

Hi all,

I'm new here, just discovered this great forum. I'm attempting to design a LAMP assay for detection of a pathogenic bacterial species. I have a couple of questions regarding the FIP and BIP primers. How are the F2 and F1c primers linked. I've seen TTTT mentioned but I can't discovered how long the TTT linker should be. Is it the dependent on the distance between the F2 and F1c primers? Can anyone recommend a good supplier of LAMP oligos in Europe. Our usual oligos providers don't have much experience with LAMP assays.

Thank you.

[dksamuel]

I want to do LAMP based detection of cucumber Mosaic Virus a RNA virus.
1. Should I d a separate cDNA step
2. can I add MuMLV or should I use AMV RT
3. will there be buffer problems between the RT enzyme and Bst polymerase
4. or is Tris-HCl- 20mM
MgSO4- 10mM
(NH4)2SO4-10mM
alone enough
I plan to do HNB and Calcein based detection
(I failed in my first experiment and so any help will be welcome)

[aahu]

Originally Posted by Cicici

Hi all,

I'm new here, just discovered this great forum. I'm attempting to design a LAMP assay for detection of a pathogenic bacterial species. I have a couple of questions regarding the FIP and BIP primers. How are the F2 and F1c primers linked. I've seen TTTT mentioned but I can't discovered how long the TTT linker should be. Is it the dependent on the distance between the F2 and F1c primers? Can anyone recommend a good supplier of LAMP oligos in Europe. Our usual oligos providers don't have much experience with LAMP assays.

Thank you.

Hi there, use "TTTT". It worked well for me. Good Luck!

[vimalpriya]

Originally Posted by msarah

I am also working on a lamp process. Did not get results with 4 primers so moved onto the 6 primer protocol and got excellent results with calcein, HNB and Mn added. Now I keep getting positive results on everything. Have remade the buffers etc with no results. Cannot think of any contamination source. Have you guys had the same problem at any point in your work?

I m a student working on LAMP . Im not getting results even with 6 primers. I designed my primers In primer explorer3 software.
I used PEG and FICOLL, then also i didnt get results.
In all reactions i got only primer dimers.
Kindly help me to get some result. If i get LAMP results i can finish half of my project.
Thank you.