PCR from a smear genomic DNA ? (View forum version)



tyrael

Posted 12 February 2009 - 07:58 AM

Hi, i jz extracted out a bacteria genomic DNA today. planning to do a PCR then.

the bacteria genomic DNA, somehow show some mild smearing in between the genomic band (upper part of gel) and RNA bands (RNA were not treated).

wonder is it okay to be PCR ? as i concern, since PCR only amplify the region i wanted, so the mild smearing part would not bring any effect onto it ? correct me if i am wrong.

Thank you

bob1

Posted 12 February 2009 - 03:27 PM

It should work, so long as there is at least one intact copy of the region that you are amplifying.

swanny

Posted 12 February 2009 - 06:02 PM

Try it and see...
Theoretically, there should be no effect.

hanming86

Posted 15 February 2009 - 08:54 AM

Go ahead with it . mild smearing is expected especially dealing with genomic DNA.

tyrael

Posted 15 February 2009 - 09:08 AM

hi all again, i worked out the PCR...
apparently, i i got the band at desired size...

however, i was SHOCKED bcoz i noticed there was a band at very very upper part !

wat's that exactly ? it appears to me as genomic DNA band bcoz it was so huge that even beyond my ladder size (ladder used : Vivantis 100bp Plus ) . how could it be possible ? bcoz DNA get denatured over and over again, and only region that flanked by primers would be amplified, by the end of PCR, i shouldnt get this kind of big size DNA..

is that the contamination problem to you all ? correct me again if i am wrong. Thank you

phage434

Posted 15 February 2009 - 01:47 PM

Any genomic DNA you put in will still be there. You may be using far too much template DNA in your reaction.