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'ladder bands' in PCR - (Apr/25/2007 )

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hi there..

Doing straight forward PCR. amplifying a 265bp fragment, i've managed to do it in the past now i'm having some issues! started getting smears but i know my DNA isn't degraded, etc. now i'm getting these strange 'ladder bands'. they literally look like i've loaded a ladder in the lane instead of PCR product! i've tried playing with the MgCl2,etc and changing my cycle but optimisation just isn't working. i really don't think its non-specific amplification. changed all my reagents, still doesn't work. a colleague is working with the same primer set and has no problems. also sterilised my pipettes but that hasn't made a difference. maybe its something really obvious but i'm out of ideas and i'm willing to try ANYTHING at this so any suggestions would be much appreciated! smile.gif

-marika-

Maybe your loading buffer is contaminated with a DNA ladder? We had that once.

-kr├╝melmonster-

thanks for the speedy response..my ladder and loading buffer have never come into contact smile.gif using the green gotaq buffer

-marika-

Where does the ladder start - at your expected product, or higher? Do the 'rungs' look like they are multimers of your product?

Try heating your primers to about 60 degrees for 5 minutes before you add them. Primers can be strange little things sometimes.

-swanny-

they start at my expected product..

i will definately try heating them to 60 degrees first, thanks so much for the suggestion!!

pleez hold thumbs smile.gif

-marika-

does that mean you are getting equal sized bands for every lane within the smear?

i was getting those for my extractions, but that was before amplification. it was weird but i found out its due to apoptotic degradation. definitely your dna its not degraded otherwise you wouldn't be getting products.

i would think it's just non-specific amplicons. or that the protocol is not optimised for those primers. do you have a control? i always run a control to help me figure out things when they go wrong.

blink.gif

-g├╝ndl-

hi there..i think i should upload a pic.. smile.gif
it's already been optimised, been working fine before now suddenly i have these weird bands. Although it is optimised and has worked before I tried to optimise it again but nothing makes the slightest difference! I PCR'd DNA samples that have amplified for this amplicon in the past and they also have the bands! Maybe i should try adding DMSO even though i'm not convinced that its non-specific i'm willing to try anything! smile.gif just put in a PCR using the primers heated to 60 degrees for 5 minutes so i'll see what happens...

-marika-

Have you tried a touchdown PCR?

-Zouden-

NO! not yet..will set one up now! heating the primers didn't make a huge difference sad.gif
no weekend for me smile.gif
once again thanks for all the suggestions!

-marika-

sigh..the touchdown PCR didn't get rid of the bands sad.gif really not loving labwork right now!!

any other suggestions? anyone? i'm kinda at my wits end!!

-marika-

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