Hi Mariel,

Doing PCR on minipreps can generate multiples band.

You can get your band of interest plus coiled and super coiled structures of your plamid that you can see even after PCR. These other bands are very high.

if that's the case, you can retry you PCR by decreasing the amount of plasmid in the mix. The intensity of the extra bands should decrease.

Best

Hi,

Gilles gives a good point regarding template concentration. Also, depending on which primers you use you can get multiple bands. I would suggest using a primer on your plasmid and another primer in your insert, so you can test both presence of a full-length insertion and its orientation. If you are going to use that primer combination often, you could set up a gradient PCR to determine the optimal annealing temperature for your primers.

Sometimes it also happens that by chance you get a band of the correct size but the insertion is not what you want. Then, after colony PCR I would recommend to do minipreps of 3 colonies to check the plasmids by restriction digest. This will tell you if the insertion is correct - think for instance of the case of three inserts ligating in tamdem as 'EcoRI-insert-NdeI-NdeI-insert-EcoRI-EcoRI-insert-NdeI'. You could clone this in a double-digested plasmid, and depending on your primer design a PCR would give you the band you want plus more bigger bands. A well-planned digestion would tell you if you have a single insert and its orientation.

Alternatively, you could skip the restriction digest and go straight away for sequencing using primers in your plasmid. This is a standard procedure before carrying on -you need to make sure that your insert has the right sequence, unless maybe if you have checked it before and you are doing just restriction-ligation or recombination.

Cheers,

Ruben

As Ruben suggested, I think restriction enzyme analysis would definitely be a better way to check your ligation than PCR.

Hi Mariel,

Did you try to put also your restricted insert on a gel? That way you can see if you get more then one band from your insert. Was the insert obtained by PCR or was it cut out of another vector?

You could also do a control PCR on the empty plasmid and see if you get additional bands.

By the way, you can also do a colony PCR on those colonies. Saves you all the mini prep work and depending on the method you use, also some money ;).

Good luck

Michaela

hi Mariel,

I wil suggest you to do restriction digestion to confirm your positive clone and you can also do at same time colony PCR or ypu take very less amount of miniprep of colonies and do PCR