(LAMP) Problems with Loop-mediated isothermal amplification

[jlamp]

Hey, this effect is due to dNTP and / or Magnesium concentration. You should adjust both.

Someone new hints for false positive? Primer or contamination?

[aahu]

Dear friends,

I'm working on LAMP for the past few months. I have few hints to solve your false positive problems.

1. NEVER use same pippetor to make master mix and to load LAMP product in agarose gel.

2. Check the position of your primers in the target gene. If your F3 and F2 primers are side by side or too near, it might results false positive. Make sure there is some distance between F3 and F2 primers. Check out some journals for the illustration of the primers position.

3. If you designed more than 1 set of primers, you can mix match your primers as suggested by someone in this thread to avoid false positive. When mix matching make sure there is some distance between each primer.

Good Luck!

[nikan]

Dear friends,
I have a question, does any body know what is wrong with making mgso4 solution to the concentration that we need insteead of buying prepared mgso4 solution. If I order mgso4 powder and make the solution myself does it cause any problem?

[nikan]

any idea?!!!!!

[sittingchair]

Hey!
there is nothing inherently wrong with doing this but precaution must be taken to measure correctly using the correct math and to be sure to use uncontaminated water

[nikan]

Originally Posted by sittingchair

Hey!
there is nothing inherently wrong with doing this but precaution must be taken to measure correctly using the correct math and to be sure to use uncontaminated water

I made it with double distilled water in sterile condition and then autoclaved it. I used the correct volum of powder and water.
I hope nothing is wrong with it.

[butters]

Generally its ok, just make sure your chemical is at least molecular grade.

If you are not careful you may introduce foreign DNA or degrade DNA.

[nikan]

Originally Posted by butters

Generally its ok, just make sure your chemical is at least molecular grade.

If you are not careful you may introduce foreign DNA or degrade DNA.

I used Magnesium sulfate heptahydrate that is for analysis.
It seems that I'm going to do a wrong job. What's your idea? What is the difference between molecular level mgso4 and this one?

[sittingchair]

As far I know, there is no problem with using this reagent. You will need to account for additional molecular weight of the 7H2O when doing your calculations, however.

[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.