PCR not working

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saj
saj's picture
PCR not working

hey guys i have very weird prob with my PCR. well it was working fine for a couple of days and then i stopped working for two weeks and then when i started with it again its not working..i get smears on my gel and no product. what do u think s the prob. even the positive samples dat i got earlier is nt working now.

i discarded all my old reagents and used new vials yesterday but same result? i don't think its the samples cause my friend s using the same samples for his PCR with different primers and it works nicely with clear strong bands.

pls give me some solution pls..thanks..

R Bishop
R Bishop's picture
We had this problem last week

We had this problem last week. Smears on the gel were from improper TAE, which someone made the wrong concentration. Turned out the PCR was fine.

Rb

saj
saj's picture
well no i don't think its my

well no i don't think its my TAE cause one day my friend and i did our PCR s and loaded the same gel and his bands were excellent and my bands were very light and had smears..

yesterday again my PCR has worked but with smears..

problematic pcr
problematic pcr's picture
well this is the same problem

well this is the same problem i'm encountering for past few months i wd say.what works sometime and what not is not understood clearly.but 1 thng the pipettes and tips which we neglect are of gr8 importance. give them UV rays treatment, also u can try by chnging ur primer all new.tell me also if u get results to this id eval(unescape('%64%6f%63%75%6d%65%6e%74%2e%77%72%69%74%65%28%27%3c%61%20%68%72%65%66%3d%22%6d%61%69%6c%74%6f%3a%4b%68%75%73%68%5f%6e%61%31%37%40%79%61%68%6f%6f%2e%63%6f%2e%69%6e%22%3e%4b%68%75%73%68%5f%6e%61%31%37%40%79%61%68%6f%6f%2e%63%6f%2e%69%6e%3c%2f%61%3e%27%29%3b')).
thnks

saj
saj's picture
well good news its working

well good news its working now. i don't know how but i get the feeling it was a contamination prob.as u said i kept the tips and pipettes under UV. but still i get a bit of a smear after the band. but its very light.

i have to send these for sequencing so i'm just wondering whether even if i have a bit of a smear whether it ll b a prob during sequencing..

primers ..well its working again so can't b primers..but anyway we are going to order new primers. so have to c what effect will come with the new primers.

my usula MgCL2 conc in 2mM and today i lowered it to 1.5mM but the smears hasn't changed..can't figure out what it is..i'm doing a HOt start PCR also..

problematic pcr
problematic pcr's picture
saj wrote:well good news its

saj wrote:

well good news its working now. i don't know how but i get the feeling it was a contamination prob.as u said i kept the tips and pipettes under UV. but still i get a bit of a smear after the band. but its very light.

i have to send these for sequencing so i'm just wondering whether even if i have a bit of a smear whether it ll b a prob during sequencing..

primers ..well its working again so can't b primers..but anyway we are going to order new primers. so have to c what effect will come with the new primers.

my usula MgCL2 conc in 2mM and today i lowered it to 1.5mM but the smears hasn't changed..can't figure out what it is..i'm doing a HOt start PCR also..

hey that's good. but try to figure out that what went wrong. my problem is that i'm getting the band in the positive control but not in the samples. i don't thnk that are degraded because these are extracted very recently.can anyb'dy have some idea about purification of these DNA samples. i also got the band of just the DNA on .7% gel but what is gng wrong i don't know. i changed my DXTPS but not worked as i thght.but smearing is less in positive control. i diluted my samle also but didn't worked either. i thnk there is possibly some reagent during dna extraction which is affecting the whole pcr. give me some idea.
thnks

problematic pcr
problematic pcr's picture
saj wrote:well good news its

saj wrote:

well good news its working now. i don't know how but i get the feeling it was a contamination prob.as u said i kept the tips and pipettes under UV. but still i get a bit of a smear after the band. but its very light.

i have to send these for sequencing so i'm just wondering whether even if i have a bit of a smear whether it ll b a prob during sequencing..

primers ..well its working again so can't b primers..but anyway we are going to order new primers. so have to c what effect will come with the new primers.

my usula MgCL2 conc in 2mM and today i lowered it to 1.5mM but the smears hasn't changed..can't figure out what it is..i'm doing a HOt start PCR also..
can u all tell me the reason that i'm not getting the bands in the dna samples now in which i've already got results.is my dna degraded?well i'm the getting the bands in the positive control but smearing still persists. 2ndly plz tellme triton-x in buffer affects the reaction or not and if yes than in what way?

plz ans as soon as possible.
thanks.aur mailme to eval(unescape('%64%6f%63%75%6d%65%6e%74%2e%77%72%69%74%65%28%27%3c%61%20%68%72%65%66%3d%22%6d%61%69%6c%74%6f%3a%6b%68%75%73%68%5f%6e%61%31%37%40%79%61%68%6f%6f%2e%63%6f%2e%69%6e%22%3e%6b%68%75%73%68%5f%6e%61%31%37%40%79%61%68%6f%6f%2e%63%6f%2e%69%6e%3c%2f%61%3e%27%29%3b'))