2 replies to this topic

[Pi341]

I have a plasmid isolated from yeast. When I amplify my desired insert fragment with two reported primers I get a weak but clear and sharp banded DNA fragment on an agarose gel. After cutting and gel extraction this fragment is re-used for further amplification. I use the same PCR method and the same reagents as befor, but I get a big smear on an agarose gel. I already tried to vary PCR conditions, including Mg2+ or DMSO treatement. Reloading the fragment form the first amplification on a gel gave a sharp band at the right position. The smear does not come from overloead, contamination etc., because a positive control gave always the desired product. I can not see where I may treate my template in a bad way.

[hula]

Could it be possible that there is no insert in your plasmid, because you said you only got a weak band whichc might have come from contamination?  How about cutting your plasmid to see if there is a correct insert?

[tltan]

Hi Pi341

Hi I read somewhere that diluting your starting product could help.

As the template is a PCRed product, the concentration is still quite high compared to the amount of template you normally add when doing PCR.

Therefore, probably, a dilution of 1:10 of the PCR product followed by loading 1ul as template would do the trick. Also, lesser amount of cycles should be used -to prevent mutation to products as well as resulting in smear like result.

Hope the above helps. Have personally not tried it but I read it somewhere in this or another forum.

Cheers,
tltan