Plasmid size should not be a factor at all. Restriction enzymes can digest large plasmids, viruses and genomes without problem. However both of these enzymes can be impacted by dam or dcm methylation.

If you have the sequence of the DNA you can scan the predicted digestion sites and determine whether they overlap a methylation site. If they do, then that is likely to be the problem. The only solution would be to prepare your plasmid DNA from a host strain that does not methylate dam or dcm sites (depending upon which one is the problem).

If that is not the issue, then it might just be a dirty DNA prep that has something inhibitory. You could just try to make another plasmid prep and try that.

Dear, Ting Chung Chau

According to the NEB product catalog, ApaI is recommended to be used at 25°C. On the other hand, XbaI is recommended to be used at 37°C.

The half-life of ApaI is stated to be 30 minutes at 37°C. Therefore, we propose the following method

First, digest with XbaI at 37°C for a sufficient time.

Next, add a sufficient amount of ApaI and digest at 25°C for a sufficient time.

Alternatively, the order can be reversed.

Anyway, the ApaI reaction should be done at 25°C.

I hope this helps you.

Narumi

Hi Ting, I think the band of the 2kb fragment will be faint relative to the 13 kb band even in a successful digestion, just because of the molarity differences. You may want to check your ligation step, as this could be the problem. Make sure to use a negative and positive control at this step. All the best.