PCR shows insert, but digestion of plasmid failed to cut out insert - (Jul/31/2006 )

Everything seemed fine to me in the cloning process. But problems popped out when I wanna amplify the cloned plasmids.

Firstly, I just use the bacterial stock I kept to amplify. I extracted plasmids using mini-prep and measured the concetration as well (300ng/ul, 260/280 = 1.4). I then double digested 1ul of the plasmid and load both undigested (1ul) and digested plasmids for gel electrophoresis. Surprisingly, there's no band for both lanes.

Secondly, I transformed the original cloned plasmid (the one which showed correct bands after double digestion) using both DH5alpha & TOP10 cells. But I only got like 100 colonies, which was abnormal as well (since there should b lots nd lots of colonies for plasmid transformation). Moreover, the colonies are very small. I picked some colonies to grow overnite but only 1 can grow. I extracted the plasmid using mini-prep and double digested as well. Same as previously, there's no band for both digested nd undigested lanes.

I know there must b something wrong with the original plasmid. But what is the prob?

This really caused me much headache since my boss is urging me everyday to settle this. And I have to solve this out this week so that I can go back to my home town. So really really hope somebody could help me check and give me some suggestions.

u may not be extracting your plasmids. they may not be eluting down the columns. what is the lenght of your construct. are you eluting it with elution buffer or with water. if using water, check that the pH is around 7-8.5
i always load at least 3 microliter out of 30ul extracted plasmid on a gel to see whether it is ok. 1 microliter may not be enough to see on a gel although you measured the concentration as 300ng/ul, spectros sometimes lie
if i were u, i would load more of the extracted plasmid on gel to see whether it is there with a low conc. or not

dodosko, thnx a lot 4 suggestions. I somehow figured out the reason.

I run gel to check my original cloned plasmids, but there are no bands. I also noticed from the gel pic tat the concentration is extremely low the time I discovered this clone. (the band is very faint on the gel pic.) Therefore, I deducted tat the cloned plasmid is very impure, so it has been degraded during these few weeks.

My collegue told me he would transform the plasmids immediately to prevent degradation occuring. Sigh! Why I transformed only till it has degraded!

Luckily 4 me, i still keep the purified insert & vector. I've ligated nd transformed. Gonna pick colonies this evening. ^^