Strategies for gene cloning and expression - Discuss various methods for it (Feb/11/2007 )

I would like to create a thread to discuss different strategies used for gene cloning and expression.

The method that I used is double cloning. First, I did TA cloning after gene amplification. After that, I did digestion for both of my cloning vector (with my gene of interest) and expression vector. Then I did sticky end ligation. For final confirmation, I sent it for sequencing. Once sequence is confirmed, I started my expression.

I am just wondering, can I just clone the gene straight away into the expression vector? Save the hassle of inserting into cloning vector and then digest it and ligate it into expression vector?

I read somewhere that I can actually do TA cloning straight away into expression vector. Is it true? How about blunt end ligation? Just 1 step cloning, it will be easier and less time consuming.

I am not complaining about how tedious about 2 steps cloning. I am actually glad I did that. At least I learned alot of stuff. But how about the other strategies? Thanks alot for those willing to discuss.

i go for ligation strategy with the fewest steps. I would PCR my insert with primers containing restriction sites. Digest the PCR product and ligate directly into the expression vector.

If the desired constructs is made of multiple inserts, I would build the construct via a single multiway ligation step on a cloning vector (eg bluescript). I would willing go as high as a 6 way ligation.

And if required, later move this multi-insert into the expression vector. Also depending on expression vector complexity, I am also willing to tear appart the expression vector and rebuild it on pBS with the insert as a single multiway ligation.

After construction (screening with PCR and later restriction enzyme), I would sequence the final plasmid product.

I would agree with perneseblue.

I routinely add sites to my PCR primers and clone it into the vector of choice. Saves a lot of time.

Using a cloning vector is definitely unnecessary, your insert can be cloned straight into the expression vector using restriction sites designed in the primers. Design the sites on the 5` end of your primers and leave about 4 bases outside the restriction site to ensure the enzyme cuts efficiently. As for blunt cloning, it is definitely worth the added effort of creating restriction sites in your primers and cutting the insert. Blunt cloning has several down falls over sticky end cloning:

1. blunt end ligations are much less efficient than sticky end ligations
2. Vector preparation is more complicated and time consuming
3. Blunt vectors produce more colonies containing no insert (background colonies)
4. It is not directional - the insert may ligate in forwards or in backwards

Good questions and good luck with your construct,

Rob

Hmm guess cloning vector isnt necessary. So, what's the point of them if I can just straight away design primer with restriction sites and clone it straight into expression vector? Could primer with addition of restriction sites will be harder to amplify the sequence?

What is the logic behind 2 steps cloning? To make sure getting correct sequence?

Thanks alot, guys.

TA cloning might help if the sites that are required for further cloning are present in the TA vector.

I havent found amplification difficult when using primers with additional restriction sites.

One needs a cloning vector, let it be a TA or any other vector such as Bluescript. Its easy to assemble different fragments in them and then clone the whole thing into the actual vector.

cloning is everytime very difficult to me and my ligation dosent works
when it works i really dont why it works.............following the same strategy i tried another sample and fail. fail and fail and success again and i donno what is the reason

Sometimes there could periods when cloning doesnt work. But trouble shooting does help in the long run. My old lab mate had a project which involved making 100 different vectors and he managed it in a year. But the initial period he did fail miserably. But he learned from his failure to complete it succesfully.

be vary of the digestion time, amount of DNA being used for ligation and use good ligase and good cells.

Good Luck with cloning !!!

My job is to clone genes and like your lab friend Scolix you learn over time what works and what doesn't. There's probably two major things I'd tell people to focus on when cloning. Firstly, choose the restriction enzyme sites well. I've posted before on how to choose good restriction sites but I think it was within another post so I'll re-post the info soon. Choosing good restriction sites in all honesty is probably the single most important factor in successful cloning I believe.

Secondly, ensure the vector is prepared well. Well prepared vector will clone almost any DNA. A few tips on that:

1. Circular vectors usually require more units of restriction enzyme (RE) to complete digestion because they are supercoiled. Supercoiled DNA is more difficult to cut than linear DNA and unit definitions usually use linear DNA (usually lambda DNA). The NEB catalogue has a list of how many extra units are required for the more common REs.

2. Always de-phosphorylate the vector prep. Even doubly-digested vectors can produce empty vectors due to either a ) undigested circularised vectors being transformed or b ) incomplete singly-digested vectors re-ligating - digestions are never 100% complete.

3. Whenever a new vector preparation is made always perform a vector-only ligation to see how many background colonies are produced. If a lot are produced, make a new vector prep. Poor vector preps that produce a lot of background colonies make you waste a lot of time screening for colonies that contain the insert and more colonies need to be screened every time.

4. If using a vector prep often, make a new prep after every 3 months. Because we clone into the same vector almost every time, we use the same vector prep quite often. After 3 months or so, it's amazing how poorly the ligations start to go. The vector becomes more difficult to clone into and clones containing the insert start to contain vector deletions on either the 5` or 3` side of the cloning site due to degradation.

5. Ensure the integrity of the circularised vector stock has been well maintained. If you have poor degraded circularised vector before you start digesting the vector, this really puts you behind the eight ball when trying to get a good vector prep. It's easy to check the integrity of the circularised vector stock you have by digesting the entire vector in even 5 or 6 positions and confirming the correct brightness and size of the bands on a gel. If it's no good, or even if you are not sure, save your self the hassle and re-transform the circularised vector stock, plate out, pick a new colony, grow it overnight and miniprep it. Sounds like a lot of precaution but totally worth it because if the stock is off it really makes a difference.