PCR trouble.....help! - (Jul/26/2006 )

Hi

kindly c the attachments of thr pcr products i have got the product suppose to be 1000bp..but i have got none in that region....someone suggest i have been in trouble since a month with this...
i have tried doing all the changes possible mgcl2 conc,,,,annealing temp.....dna template conc.... nothing giving effect..please help me......

thankyou

Can we please know the size of the bands we see in the marker? That would help...

have you titrated your primers? or, tried a new batch of primers? new batch of DNA? diluted or not, your prep could be degraded...

it looks as though the gel on the far right may have primer-dimer; I agree with DNA Factory...an idea of standard band size would help

and what is the nature of your primers? have you added linkers or RE sites to the end, meaning you would have to jimmy the annealing temp to get good product? (I only ask because this is a common mistake, and I got busted with it myself )

please give us more information

That looks a lot like a 100bp ladder, so each would be 100bp (I could be wrong). I'm having problem with same result, nothing show, and streaks all the way =( Could my DNA be degraded? I keep them in -4C fridge, I know in some lab ppl kept them at -20C

Hi all...

The DNA ladder that i used is 1kb ladder..500 from below....500,1000....like that...
I tried to check for even primer dimer formation by juz running the primers without adding the template but in that gel i got no streaks nor band to saying there was nothing.....
iam unable to trace what could be the problem for streaks....and not even non specicif bands...doing in all sterile conditions....the primers showed no problem when designed in gene tool....it after that we had ordered for it....and now this comes up...
tell u the pcr conditions....
step1- 95°c - 5min
step2- 94°c - 20sec
step3- 50-65 -30sec(annealing temp)
step4- 68°c - 2min
step5- go to step 2- repeat 34 times
step 6- 68°c - 10min
step7 - halt at 4°c

I have been doing the pcr in gradient where i had set it for a series of temperatures(50-65°) but still dont get. tried to change the conc of DNA from 25-400ng....but still no results...
have hung for quite a while without knowing what more can i do.....
had designed the primers based on the DNA sequence given in the gene bank and had seen fro dimer ,loop formation etc only after confirming these things we had ordered for primers.....
so now what?????

Hi...

suggest me something please.....
what more info is required?????

Thank you

hi
After reading your message one thing came to my mind that are u getting the same result in the -ve control also? and what is the Tm of your primers, and length of the primer is your both primer is having Tm in 50-65 c range? do u have any +ve control?
check the tm once again and keep for 1 min for annealing and 1 min for denaturation ie 2nd step.and try at low temp by gradient pcr.ok

Just to make sure your template is good, run a positive control.

The template has no probs its without shearing....and has got a gud od(1.77) without protein contamination......i have tried with gradient at diff annealing temp and time now giving an annealing time of 3min but no positive results.....what else can be done???? the primer have tm of forward-61 and reverse-63.....and they are 26-27bp primers.....
what more can i change to get results??????

Just as it is already mentioned you should use a positive control to find out what is wrong.

I had the same problem a few months ago and even the positive control failed. I tested different polymerases, too, but finally it was the dNTP-mix that didn't work... after preparing a new one everything was fine.