I am currently trying to clone a 2.75 kb cDNA into a 5.15 kb long cut pBABEpuro cut vector. This is a conventional single cut restriction cloning. I obtain my inserts by making site directed mutagenesis on a template cDNA (without mutagenesis kit). I have to make various constructs for various mutations. Only one of them barely worked, 36 colonies were screened by colony PCR and only one colony gave positive result. For most other insert preparations, I get no positive colony. Have not tried screening more than 20 colonies for each construct.
I have been doing trouble shooting experiments for a few weeks:
> When I transform the allegedly cut vector, it gives colonies (approximately 1 colony per nanograms of cut vector). My competent cells give zero colony in negative controls and completely fills the plate with colonies with 1 ng vector as positive control.
> I was first suspicious about incomplete digestion. Directly following the manual of the enzyme, I repeated extended digestions, up to overnight at 37 degrees. Results were the same.
> I then got suspicious about possible plasmid contamination. I checked every reagent with nanodrop that is involved in my molecular cloning experiments. Results were not meaningful. Then I transformed my reagents directly to see the presence of detectable plasmid DNA that can result in colonies. I did not observe any colonies in all of them. These contamination tests used kit buffers, enzyme buffers (endonuclease, phosphatase, ligase), the double distilled water that I use for my reactions, and even heat inactivated enzymes, the stock glycerol solutions that I store my frozen bacteria in, LB media etc..
> Then I gave up on the lab bacteria stock. Retransformed the vector, pick a single clone, made prep out of it using miniprep kit. Identified it with restriction mapping. Results were the same that I still got colonies possibly due to uncut colonies.
> I picked these unwanted colonies, minicultured and made quick&dirty preps out of them. Restriction patterns indicated foreign DNA. This signals contamination. I can not get over this given that I have refreshed everything, including plastic consumables etc, cleaning the pipette sets, I can not do anything on top of it. I decided to ignore it.
> I ran into another problem when I saw that the vector was inefficiently dephosphorylated. I made another control experiment in wihich one plate I directly transformed phosphatase treated cut DNA, and in the other I plated bacteria that was transformed with the same DNA prep with same quantity, but treated with ligase in the absence of insert. The latter one gave 4 folds of colonies when compared to the other plate. The increase of colonies corresponds to the increase when I tried to ligate my cDNAs, which explains a lot.
> I can ignore uncut foreign vector contamination, but I can not ignore insufficient dephosphorylation. Because vectors always favor ligating in on themselves. Manipulating vector/insert ratio, heating and gradually cooling vector/insert mixture for sticky end annealing etc. nothing did wonders.
> I am about to compare the efficiencies of my labs nucleotide modifying enzymes to those of other labs with simple control experiments as the ultimate step of my troubleshooting adventures. But as I can see the end of this, there are not many ways out:
· A new set of enzymes can be ordered.
· Everything can be done under its optimal conditions. For example, DNA prepartes can be eluted from columns with water instead of TE. So that the only salts that would be present in the reaction mixture would be those of enzyme buffers.
· Amount of DNA can be sacrifed in the favor of obtaining clearer DNA after each enzymatic reaction and for buffer exchange, doing subsequent cleanups after each reaction.
· But none of these should be essential because these enzymes are stated to be working in each others buffers, by the company.
> Use of site directed mutagenesis kit is my last hope as it will give zero background (Unless Dpn1 decides not to work). But it is my last hope because it is too expensive and I do not like the idea of amplifying whole vector in vitro instead of using the bacteria as the system, no matter how high fidelity the kit it is. Because I can not afford sequencing the whole vector. But in restriction cloning, I will only sequence the cDNA.
Can you give any suggestions based on your experience. Have you encountered such things and how did you get over them if yes? Thank you for your patience.