SMear instead of PCR product - (Apr/03/2006 )
I'm having smear instead of PCR product.
Could it be RNA?
And if this is RNA, at what step should I add RNAse?- after DNA extraction when resuspending DNA in H2O or TE?
Thanks!!
-yata-
hi
if the smear is below 100pb it's primer aggregation and means your pcr didn't functioned well
If it's upper, it's a pcr problem.
if you think it's RNA, add RNAse after resuspendng DNA in TE. (is it a kit or a alcaline lysis method you use?)
-fred_33-
1st, that smear was in only in 1 sample, and was of every size.
Then, I decreased annealing temp by 2 degrees, and the smear became <0.5kb
I'm using all kinds of extractions, including alk.lysis.
Thanks!! 
QUOTE (fred_33 @ Apr 4 2006, 04:16 AM)
hi
if the smear is below 100pb it's primer aggregation and means your pcr didn't functioned well
If it's upper, it's a pcr problem.
if you think it's RNA, add RNAse after resuspendng DNA in TE. (is it a kit or a alcaline lysis method you use?)
if the smear is below 100pb it's primer aggregation and means your pcr didn't functioned well
If it's upper, it's a pcr problem.
if you think it's RNA, add RNAse after resuspendng DNA in TE. (is it a kit or a alcaline lysis method you use?)
-yata-
HI
Well if it's a global smear, it's not DNA. how does your template look like? Can you gel test it?
Did you try DMSO addition?
-fred_33-