Unstable clone? Options

[j6yan]

Hey Guys,

I'm having a really difficult time cloning in this insert. Everything is good up until the transformation step. I get colonies on my transformed plate compared to my control. However, when I go about to selecting colonies and screening I don't get any positives, not even any potential negatives, they are negatives that shouldn't even be there. I end up getting banding patterns that are not right at all after I digest with RE.

Is it possible there's recombination going on? I sometimes get bands that are only 1-2 kb in size and my vector itself is 5kb. My insert is 3kb. I don't even know where these bands are coming from. I've tried so many different ways and so many different times with new reagents, new plasmids, new vectors so I know its not contamination.

I've been doing this for so long and still no success. We think our gene is just very unstable since the protein structure itself contains 6 repeats of a certain domain. We think this may have an affect on the gene recombining in the host.

Any help or input would be greatly appreciated!

[eldon]

Hey Guys,

I'm having a really difficult time cloning in this insert. Everything is good up until the transformation step. I get colonies on my transformed plate compared to my control. However, when I go about to selecting colonies and screening I don't get any positives, not even any potential negatives, they are negatives that shouldn't even be there. I end up getting banding patterns that are not right at all after I digest with RE.

Is it possible there's recombination going on? I sometimes get bands that are only 1-2 kb in size and my vector itself is 5kb. My insert is 3kb. I don't even know where these bands are coming from. I've tried so many different ways and so many different times with new reagents, new plasmids, new vectors so I know its not contamination.

I've been doing this for so long and still no success. We think our gene is just very unstable since the protein structure itself contains 6 repeats of a certain domain. We think this may have an affect on the gene recombining in the host.

Any help or input would be greatly appreciated!

Sounds like the sequence you're trying to propagate is unstable. Could be due to the repeats or low complexity (e.g., AT or GC bias).

SURE cells are really good for cloning unstable DNA. What I find also works is to do the transformation recovery, grow the cells once plated at room temperature and reduce the ampicillin in your plates by half. Growth of colonies and prepping DNA takes longer, but it works with huntingtin which is 10kb in size and has a very large polyQ domain.

This post has been edited by eldon: Nov 4 2009, 10:09 AM