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Bands appearing out of no where?!


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#1 hellohello

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Posted 05 November 2009 - 12:29 AM

Hi

I was wondering if anyone can help or offer some advice

Im currently doing my final year project and have hit a wall

I have perform PCR on my samples not expecting any bands except for my positive control.

After running a gel, my positive control showed a band exactly where it was expected but all my samples had bands also!!
I am almost certain that with these primer there shouldnt be any banding at all.
Furthermore the bands do not match with my positive... they are about 50-bp higher and uniform for all my samples.

i then incresed my annealing temperature and these bands still come up!!!

i dont know whether this is a pimer issue or not?


Can anyone help? Please?

#2 Vini

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Posted 05 November 2009 - 01:11 AM

Hi

I was wondering if anyone can help or offer some advice

Im currently doing my final year project and have hit a wall

I have perform PCR on my samples not expecting any bands except for my positive control.

After running a gel, my positive control showed a band exactly where it was expected but all my samples had bands also!!
I am almost certain that with these primer there shouldnt be any banding at all.
Furthermore the bands do not match with my positive... they are about 50-bp higher and uniform for all my samples.

i then incresed my annealing temperature and these bands still come up!!!

i dont know whether this is a pimer issue or not?


Can anyone help? Please?




prima facie, looks like a primer problem...maybe u can fill in us with more details....wat r u amplifying, positive, negative controls???

Edited by DRN, 05 November 2009 - 01:13 AM.


#3 hellohello

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Posted 05 November 2009 - 01:26 AM

Im analyzing my e.coli isolates to see if are pathogenic strains

for this particular case ive use STX1 primers with the PCR program:

Initial denaturation of 94C for 3 minutes, followed by 35 cycles of 94C for 15 seconds, 50C for 30 seconds, 72C for 30 seconds and a final cycle of 72C for 5 minutes

then i increased the annealing 60C .. got the same result

the positive i used was an e.coli strain known have pathogenic characteristics (e.coli933)

my negative came out perfectly fine with no bands


to further prove that my samples were'nt a pathogenic strain i ran several PCR amplications using STX2, LT, Eae, STa and STb all known to code for some kind of pathogenic factor... and found no bands present in my sample

#4 Vini

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Posted 05 November 2009 - 03:31 AM

Im analyzing my e.coli isolates to see if are pathogenic strains

for this particular case ive use STX1 primers with the PCR program:

Initial denaturation of 94C for 3 minutes, followed by 35 cycles of 94C for 15 seconds, 50C for 30 seconds, 72C for 30 seconds and a final cycle of 72C for 5 minutes

then i increased the annealing 60C .. got the same result

the positive i used was an e.coli strain known have pathogenic characteristics (e.coli933)

my negative came out perfectly fine with no bands


to further prove that my samples were'nt a pathogenic strain i ran several PCR amplications using STX2, LT, Eae, STa and STb all known to code for some kind of pathogenic factor... and found no bands present in my sample


I am sorry, but I hv got utterly confused!!! i thought u earlier mentioned that ur negative control also showed bands, but 50 bp higher that the expected one for the positive ones......in ur second post, u r saying dat ur negative control is perfectly fine..... :o

#5 hellohello

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Posted 05 November 2009 - 03:43 AM

sorry forgot to mention about the negative in the first post :o

#6 Vini

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Posted 05 November 2009 - 03:51 AM

sorry forgot to mention about the negative in the first post :o




oops!!! now i get ur problem.... hv u repeated the experiment????....was just wondering whether u hv conatminated ur samples and also do a blast check of ur primers (if you haven't already) to c that they are specific....




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