Restriction Digest Question - (Dec/18/2013 )
I am digesting a 12kb plasmid that I have constructed with NdeI. I THOUGHT the plasmid had 3 NdeI sites and as a result would yield 3 bands totaling 12kb. I will be sequencing the plasmid to make sure I have the correct sequence. But what I observed is two bands totaling 12kb. So, it seems I have two NdeI sites and the third site has mutations. My question is:
Is it possible for there to be 3 NdeI sites, but only have 2 of them cut efficiently? In this scenario all of the enzyme would bind the 2 "efficient" sites and the 3rd inefficient site would be left uncut. I am just trying to see if anyone has observed this. I am sequencing the 12kb plasmid to find out definitively, but I really hope there are not mutations, as it took a lot of work to construct this, and it feels like a slap to the face when I have to correct mutations.
The digest conditions were
1uL NdeI
1.5ug plasmid
2uL 10X buffer
H2O to 20uL
It seems unlikely. It appears that Ndel doesn't show any kind of methylation sensitivity, so that can be ruled out. Did you construct a portion of your plasmid through a Ndel site. If so, are your RE product sizes unique enough where you could narrow down which site is not getting cut?
Like you said, I think your best bet would be to sequence your plasmid and see what the heck is going on.
Keep us updated. I have never encountered this, but it would be good to know for future reference.
Im still waiting on the sequencing. The banding pattern was unique, so I do know exactly which site is responsible for this. I just find it odd that 1. I got this mutation using only high-fidelity polymerases and 2. What are the odds that it would mutate the restriction site that I randomly chose to check the insert!?
It's very unlikely. One possibility is that your final desired product is toxic, and you are selecting for non-toxic variants.
phage434 on Fri Dec 20 22:36:53 2013 said:
It's very unlikely. One possibility is that your final desired product is toxic, and you are selecting for non-toxic variants.
You are likely correct. I have noticed that the only colonies growing after 24 hours at 37C have incorrect sequences and I am selecting for them. Thus, my insert is probably toxic to the cells. I have had some success with a similar construct growing at 30C for 48 hours. I will try that to see if I can select for the correct clone.