I have an addgene vector that I linearized with a restriction enzyme and gel purified. I also PCR amplified my insert with the "tags" that were described in the addgene protocol. I gel purified my insert as well. I ran the purified vector and insert on a gel and it is pure and the correct size. I then followed the LIC protocol using the T4 DNA polymerase (EMD 70099), which was as follows:
Treat gel purified vector:
10 ul gel purified vector (8.2 ng/ul)
2 ul dGTP (25 mM stock)
2 ul T4 DNA pol 10x buffer
1 ul 100 mM DTT
0.4 ul T4 DNA pol
4.6 ul H2O
Thermocycler: 22 C for 30 min, 75 C for 20 min
Treat gel purified PCR product
10 ul gel purified PCR product (10.1 ng/ul)
2 ul dCTP (25 mM stock)
2 ul T4 DNA pol 10x buffer
1 ul 100 mM DTT
0.4 ul T4 DNA pol
4.6 ul H2O
Thermocycler: 22 C for 30 min, 75 C for 20 min
Combine & Transform
Combine 2 ul of vector and 2 ul of PCR product in total 10 ul volume (I used water) and incubate at RT for 10 min to anneal.
Transformed BL21 E. coli cells (Sigma CMC0016) according to the protocol available online.
I had a positive and negative control for my transformation. My positive control worked, but I had no colonies for the plasmid I am trying to clone. I tried extending the annealing time to 30 min and still nothing.
Can anyone suggest troubleshooting?
How long is the overlap on each side and what vector : insert molar ratio did you use?
Longer overlap will increase the possibility of recombination. In addition, you might want to use RecA. See the attached paper for more information (and an improved, sequence independent way of doing LIC).
Article Harnessing homologous recombination in vitro to generate rec...
It is possible to combine two overlapping fragments without doing any enzymatic treatment at all. They need to have at least 18 bases of overlap. Simply co-transform them by heat shock into very highly efficient chemically competent cells. (Electrocompetent cells do not work, for unknown reasons).
Lucigen has described this technique on our website ("Expresso Cloning"). It seems too simple, but it does actually work, even in RecA- cells.
Also, I agree with the advice about avoiding any exposure to UV light that is less that 360 nm. I prefer using dyes that don't require UV at all, such as Midori Green.
Thank you for the suggestions. I decided to try again and increase my insert to vector ratio (3:1). I followed my protocol exactly as before but used 150 ng of insert and 50 ng of vector. I electroporated into top10 cells this time (our lab has a large stock). I did a positive and negative control plate again.
This time, my plate had a handful of clones. My negative control had none and my positive was a carpet of bacteria. With restriction enzyme cloning using sticky ends, if I only got a few colonies, I would think it didn't work. What about with LIC? Is this method of cloning very inefficient?
Also I didn't do a vector only control plate because I didn't think the blunt ends of the vector would reanneal. Is my assumption true?
I picked three colonies and will send them for analysis anyway, but I would appreciate suggestions and thoughts.
Hi Khatera...
Do you have any news about your LIC protocol? Did you improve the efficiency?.
Best,
I should point out that 150ng insert to 50ng of vector is likely a protocol error - the ratio should be a 3:1 *molar* ratio, not by mass.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology