Hi, Ratnapal, perhaps, you need to optimized your PCR conditions, ea. salt concentration, annealing temperature etc. You can check also your solution for DNA preparation, may be one of them was contaminated. I hope this will help.

Good luck!. .

Did you blast the sequence? Maybe it is not as specific as you think? Also, as mentioned above, optimization helps, for salt concentrations and annealing temps. was the band at the correct anticipated size?

I think you need to increase the annealing temperature of the pcr condition. In this case you are increasing the specificity of the primer binding to the template DNA to avoid the unspecific binding.

Check also whether you have cross well contamination. This is a bit hard to troubleshoot if your sample loading is not careful enough to avoid the (cross) flow via submerging running buffer.

u can treat ur mastermix with uv light for 10 to 15 minutes before adding the templet DNA. this works very well.

Hi, Ratnapal, if in the control you have bands with the same size as in positive lines, you should check contamination of your isolation buffers. Try to prepare new buffers, autoclave all your homogenization staff (mortars, pestles etc), autoclave all buffers you have used for DNA isolation (but prepare new). Clean your pipets. PCR is very sensitive, if you have even track amount of "positive" DNA, you will have amplifications. Good luck!

Dear Ratnapal, other colleagues have explained earlier the possible origin of this false amplification. You can check your solutions for DNA preparation, prepare new buffers, clean pipettes etc. Moreover after that, may be to change also the conditions of PCR temperature, emphasising in the annealing conditions. Good luck!

Hi Ratnapal,1, try to increase annealing temperature; 2, try to use the solution of the cDNA preparation for your water control; If it is still there, try to cut the band and sequence it see what it is. Good luck.

Hi

I think it is better to increase the annealing temperature of the PCR as soon as possible.

HI Ratnapal,

I think you've already got a lot of really good tips/advice here.

On the very practical, basic side, here's things that come to me, that I hope can help, too. If these thoughts are too basic, please accept my apologies for stating things that are already obvious to you.

As silly as this sometimes sounds, you might also be aerosolizing your kanamycin-positive samples as you're loading into plate. I typically work with qPCR DNA detection, where we commonly only have 20-100 copies per reaction for our positive samples, and so cross-contamination is a real game-ender for us. Some things I do that consistently minimize this risk to zero:

1) dispense all of your PCR reactions intoall of the strips/wells in two steps, instead of pre-mixing your PCR reaction mix with samples, then loading. I do this by first adding reaction mix (sans samples and controls) to my *entire* strip/plate first, then adding the controls/DNA samples to each well, by carefully injecting/pipeting directly into the mixture, which takes aerosol to zero (for all intents);

2) Load samples into the reaction wells on your plate in the following order: water wells first, DNA negative controls next, and positive samples last. (least chance to "carry" positive DNA over to control wells, this way)

3) Be sure you always change pipet tips, between every well you pipet, and for every different sol being added (i.e. each reaction well is minimum two tips, one for reaction mix, one for sample).

Another concern for cross-contamination, proximity of wells to each other, can be overcome by leaving one well, or row of wells, between your waters/controls/samples.

And, if you are using plates, you *can never* be too thorough in pressing the seal down onto the top of your plates.

just an additional thought . .

how many negative control samples did you try? if it is only one, try using more control samples, it is possible that contaminations is there template DNA.

do write back if you solve your problem.

All the best