I am trying to ligate my insert of 1kbp into a vector of 4-5kbp but I am having some trouble. Here are the steps that I follow:
For the insert:
Using NotI I digest my insert out of another plasmid, run the digested product on a gel and I see two bands where expected. I cut the band with my insert, gel purified (Qiagen) and put in -20C until needed.
For the plasmid:
Using NotI I digested the plasmid, run the digested product on a gel, an see only one fairly clean band where expected. There are no smears only a tiny "tail" on each edge of the band (probably due to too much DNA?). I cut the band, gel purified (Qiagen), and put in -20C.
The first time I did this I did not dephosphorylate my vector and none of the colonies I screened had the insert.
This time I couldn't dephosphorylate after gel purification due to time constraints so I threw it into -20C hoping it would not re-ligate if frozen. I plan to treat my vector with CIP tomorrow morning. Would I encounter problems during ligation because I waited 12-18hrs to dephosphorylate my vector?
During my first try, both my insert and vector after gel purification had a 260/280 of 1.8-1.9 but the 260/230 was <1.0 (I think mostly likely due to salts and not ethanol). I was told to ignore the 260/230 ratio and go ahead with the ligation. Could these salts cause problem? I know I end up diluting the salt with H2O and ligation buffer but I have no experience with cloning and this is becoming frustrating.
Also, would it be worth using CIP/AP/SAP that has expired? I think the SAP I found expired a decade ago, AP expired 4 years ago and there's no expiration date for CIP.
Edited by Wek, 22 April 2015 - 08:10 PM.