gel purification for PCR product - (May/22/2007 )
Hi all, iam new PhD student i just started before 4 months, Im not experienced in this field could any body please help me???
Iamplified 1 kb product then i purify it by using novagen spin prep gel DNA kit, when i run 2 ul to check my purification i got my 1 kb band and another larger band is it contamination ????
I'll be really appreciated if you help me
sounds like contamination to me... try re-purifying? It shouldn't be concatomers as this is amplimer direct from PCR right? shouldn't be degradation cause it's bigger...
maybe it is there the whole time and you concentrated it?(sometimes seperation isn't complete on the gel but usually I thought it is small stuff that gets caught with big stuff (runs higher than it should) not the other way around...) I don't know the steps for your gel kit so I cannot determine if this is a likely reason or not...
ummm if it doesn't go away next time you purify or if it increases in amount then check your buffers etc for some contamination
HTH and good luck
HTH and good luck
first of all thank you very much for the fast reply, i totally agree with you about the big stuff issue this is why iam so confused
but coul you please explain more about the possibility of being there and i concentrated it????
so it depends on your isolation technique post your protocol in brief and I can answer better...
what do you mean by isolation technique???
I combined 3 wells then i loaded 150 ul of my PCR product then i cut the gel slice under UV, put into 2 1.5 tubes then i added agarose melting buffer (300ul for 1oo mg gel), incubate for 10 minute , made sure that the gel melted, then dispense 700ul from the melted gel into the filters, centrifuge for 30 sec then add 650 ul buffer contains ethanol centerfuge for 30 sec and discarded the flow through, and cenerfuge agian for 1 min to completely eliminate ethanol , then i added 20 ul prewaremed elution buffer
ok so you definately concentrated by going from 150ul total volume to 20ul total volume... that is what I meant... so you may be able to see a band that was there in the first place just too dilute to see...