Controls are obviously extremely important when setting up experiments. Without them, meaningful interpretation of the experimental results can be impossible.
I say obviously, but in my previous job as a technical services scientist, you’d be surprised at how often I found myself talking to customers about the importance of controls.
One customer commented, during our discussion over TA cloning problems, that a transformation control “…is just a waste of everything”
But in reality, the opposite is true. There is no simple way to know how good the cells are, than by doing a transformation control. And while an empty plate with no white colonies after an entire day of work is frustrating, having no idea what went wrong makes the experiment a complete waste.
So, where do you start with the controls?
1. Vector only ligation control: Often with TA cloning, people worry about vector ligating back on itself without an insert. If you are using blue-white screening, it is possible to see few white colonies with the vector only control.
However, it should not be more that 1-2% of the total number of colonies on the plate.
A vector only control (vector + ligase but no insert) gives you an idea of the kind of background you can expect to see on your experimental plate.
2. Transformation control: Every time a new vial of bacterial cells is removed from the freezer, it is good practice to perform a transformation control using a known amount of uncut vector.
The transformation control helps to show whether:
- The bacterial cells are competent
- Plates are fresh enough to allow growth of the bugs.
- You did everything right with regards to the transformation procedure itself.
3. Other Positive Control to consider: If you are really enthusiastic about having more controls, you could include a ligase control, using an insert and vector combination that worked in the past, to make sure that the ligase you are using is working well.
If your ligations are not going well, here are a few hints that might help:
1.Thaw the ligase buffer completely and mix well before using. White precipitates in the buffer could mean that one of the buffer components is precipitating out. 2 min incubation in 37C bath may help it go back into solution.
2.Multiple freeze thaws on the competent cells can impair their ability to stay competent. If possible aliquot them to volumes just enough for 2 transformations.
3.Prepare plate fresh if possible. Plates should not be stored more than a week at 4C. Do not add antibiotics when the liquid agar is hot. Warm the plates at 37C for at least 30 min before plating.
If you are only getting blue colonies on the plate…
Here are a few possible causes that most people do not consider:
1.Insert is too small to cause inactivation of the lacZ gene.
2.The insert is in-frame with the lacZ gene.
In the above two cases, you may see colonies that are light blue in comparison to some darker colonies. This just means that even though the cloning procedure worked, the insert was not the right size or of the right frame to result in a white colony. If the light blue colonies are screened, you may be lucky enough to get the clone.
3.The insert is toxic to the bacterial cell. Growing cells at 30C instead of 37C may help to some extent.
Hopefully these tips will help but if not you could take a look at our articles on pinpointing ligation problems (which provides some more details on ligation controls) and making competent E.coli (which includes a section on cell quality control).
Any questions or tips of your own? Drop me a comment!
Shoba,
A very nice and well written article. Your tips for controls are dead on. My one tip that I would add in to use several concentrations of the ligation reaction for transformation. I’ve seen many folks just include 1 amount of the ligation reaction. If you are thawing out cells, try a dilution or even 10-fold of the suggested amount of the ligation. Whats one more plate and vial of competent cells compared to repeating the PCR/ligation/transformation the next day or the next week if you did it on a thursday ;).
Rusty
I think vector only controls are particularly important as you highlight Shoba – esp as the vectors often seem to lose their ‘T’ overhang and will therefore happily blunt-end ligate.
A couple of of other suggestions for TA cloning – a higher proportion of positives was published when using a longer final extension time (30 minutes). There is also the 5′ primer sequence to consider as to whether the 3′A will be added opposite the primer (so-called PIG tailing in Biotechniques many years ago).
Finally, I fully agree with Rusty. I used to use a fixed amount of vector (very small amount) and use a few different volumes of insert.
Cheers
John
John and Rusty,
Thanks for the comments. Using a longer final extension after PCR is a great suggestion John. Taq doesn’t always add an A at the 3′ end. It will definitely help to do an extra final extension after the PCR reaction.
A few extra dilutions of the ligation reaction for transformation is worth a try considering the lost weeks/days if that one dilution does not work. Thanks Rusty.
Regards,
Shoba
i have done ligation and transformation but end up having very2 little white colonies..ist possible that little white colonies would be the desire product that i want
Hi Darul,
I would pick the colonies and set them up for an overnight culture and do a miniprep the next day. If they have the desired clones, then, you got lucky :). Usually, if ligation worked, there should be a lot more white colonies. It sometimes happens for the reasons described in the post, that you don’t have very many. In that case, the two little ones you see may very well be the right ones.
There could be 2 other reasons for the apprearance of smaller colonies.
1. Sometimes, smaller colonies could grow very late into the 37C incubation stage and are located very close to other larger colonies. Larger colonies deplete the antibiotic in the surrounding regions of the plate giving rise to smaller antibiotic resistant colonies. These, when inoculated into fresh liquid culture with antibiotic will not grow.
2. Simply contamination from other sources.
All above suggestions include everything from technical point of view but i would like to add from my personal experience that sometimes ligating direct pcr product also helps in loosing A-Overhangs.
Best of luck
Hi Ram,
Thanks for posting that tip!
Shoba
Hi Shoba, nice article.
About storing the plates at +4 degrees, I have actually been able to store my plates wrapped in polythene for up to a month and the plates, with antibiotics, still work very well.
I agree with you Fumoki that plates with antibiotics can be stored long term. Different drugs have different half-lives in plate media and some may have to be stored in the dark in addition to being kept refrigerated.
The least stable drug I know of is Zeocin and Zeo plates can be stored for up to 5 weeks I’ve found (at least that’s the longest a sleeve of plates has lasted me). In my lab, we regularly use LB+ampiciliin and LB+kanamycin plates that are up to a year old (sometimes slightly over a year old) without problems. I’ve just started using chloramphenicol plates and expect those to last at least month too.
At any rate, if background is an issue, it should be simple to replica plate on to fresh plates and identify the true drug resistant transformants.