Bands appearing out of no where?! - (Nov/05/2009 )
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?
hellohello on Nov 5 2009, 02:59 PM said:
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???
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 94°C for 3 minutes, followed by 35 cycles of 94°C for 15 seconds, 50°C for 30 seconds, 72°C for 30 seconds and a final cycle of 72°C for 5 minutes
then i increased the annealing 60°C .. 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
hellohello on Nov 5 2009, 02:56 PM said:
for this particular case ive use STX1 primers with the PCR program:
Initial denaturation of 94°C for 3 minutes, followed by 35 cycles of 94°C for 15 seconds, 50°C for 30 seconds, 72°C for 30 seconds and a final cycle of 72°C for 5 minutes
then i increased the annealing 60°C .. 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.....
sorry forgot to mention about the negative in the first post 
hellohello on Nov 5 2009, 05:13 PM said:
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....