7 replies to this topic

[KhaleesiDany]

greetings 

 

im trying to ligate a 555bp insert into a 6kb vector both plasmids were digested with pci I to extract back bone n insert of interest... i did dephosphoylation of backbone, n ligated at 22 degrees celcius overnight, the next day i transformed half the ligation mix n plated...the other half i left at room temp to transform the next day incase the first didnt work, n the following day i had no colonies.. my positive control plate had a few colonies, 1 of my experimental plates (A) had four another ( B  )  had alot the others non,, i took 10 from plate b and all four from a, after overnight inoculation of plate b colonies nothing had grown as the solution of LB was still clear.. i did have trouble picking colonies from plate b because they had small colonies n we use a pipette tip which i think sometimes digs the colony into the plate rather then lifting it up. the four colonies from plate a grew n i mini prepped n digested but the bands were not of correct size they were not even the sizes of a religated backbone, i dont know what thy couldve been... i transformed the remaining ligation mixture but the next day i had no colonies on any plates...how do i better my ligation protocol?... i used kapa ligase kit, i even used thermo t4 kit, i used a 1:3 ratio of vector insert.. i did have very low dna concentrations of vector n insert though.. so i also tried adding a lot more insert because the starting concentration was low.  

Edited by KhaleesiDany, 23 January 2015 - 01:45 AM.

[CPRES]

There are so many things to point out, so I wouldn't. But as you say " i did have very low dna concentrations of vector n insert", I think that could be the reason. YOu should at least have 50-100 ng of linearized, purified plasmid in the ligation reaction. That is crucial. Then comes the 1:3-1:5 molecular ratio of vector:insert. So in your case you need 100 ng vector and 25 ng of insert in your reaction. Or 50+12.5. But don't go below 50 ng for linearized vector.

[neuropath]

Hi KhaleesiDany,

 

Sounds like you are facing those teething problems that often plague people who just started out doing cloning. When you read about it in protocols or articles, it sounds very simple but actually, there are many things to look out for.

 

For most T4 ligases, you only incubate at room temp if you are doing a quick ligation, say for 1-2 hours. For overnight ligation, most protocols suggest 16 deg C. I just incubate at 4 deg C and it has worked so far

 

It is not advisable to store DNA at room temp for any length of time. This is especially important for ligation reactions because the amount of DNA is usually small. I either store them at 4 deg C for short term or -20 for long term.

 

One thing that many people overlooked as an important factor is the ligation buffer. It contains ATP, which donates the phosphate group for the ligation reaction.....and ATP is heat sensitive. If you leave your buffer out for a long time at room temp or worse, thaw it in your hands or at 37 deg C, the ATP will degrade and your ligation will not work.

 

Those colonies on plate B are probably not bacteria. If they are a very opaque white with a very sharp outline that sometimes makes a depression in the agar, they are most likely fungus. They will grow very slowly in overnight liquid culture or will form a stringy growth if you look closely. To avoid fungus, you must practice good aseptic techniques e.g. pour plates in a clean environment, make sure your water is clean, use autoclaved tips and not leave tip boxes open etc.

 

Another important factor is your competent bacteria. If you had used circular plasmid as your positive control and only got a few colonies, then I would start suspecting the quality of your competent cells. Even a few nanograms of circular plasmids should give a lawn of bacteria if your cells are good. Get new cells or borrow some that you know for sure had worked for others 

 

Good luck.

 

~neuropath~

[labtastic]

Room temp overnight ligations work great. Been doing it for years, 100+ colonies when the upstream cloning steps are efficient. Closed circular plasmid DNA is very stable (assuming you don't have DNAse contamination).

 

Agreed the ligase buffer can go bad easily. Freeze thaw cycles are what will do it in the most. ATP isn't that temperature sensitive (after all it works in PCR's at 95C right?), but in general if there is any doubt on the status of the ligase buffer, grab a fresh tube, aliquot and store in a reliable non-self defrost -20C freezer.

 

Also agreed that probably the biggest bottle neck with low-efficiency transformations like from ligations is the competency of your cells. If your chemically competent cells are homemade, make sure they are made by someone who knows what they are doing. Cells made half-assed will give crap for results. If there is any doubt, buy commercially prepared cells, or transform by electroporation. Making electrocompetent cells is easy and less sensitive (grow to mid-log phase, harvest, 3 washes in 10% glycerol, aliquot and flash freeze), but cuvettes aren't too cheap when making dozens of constructs.

 

Also make sure you antibiotic is correct, as well as its concentration (sorry had to state the obvious...only because I've made that mistake).

Edited by labtastic, 05 February 2015 - 12:58 PM.

[phage434]

Room termperature ligations work fine in 10 minutes, no need for overnight (unless you need to go home to dinner). "Ligation" problems are almost always problems with either the DNA amount or quality, or the competent cells. Second the above competent cell discussion. If in doubt, measure the competence by transforming 100 pg of a plasmid such as pUC19, doing serial dilutions of the cells, and calculating the CFU/ug of plasmid. by counting colonies. You need at least 10^7 cfu/ug to do cloning. 10^8-10^9 is better, and easy with commercial cells.

DNA quality is also very important. You already are asking for problems by doing dephosphorylation of the vector. Instead, I'd highly recommend rethinking your cloning strategy to use two different enzymes which can be heat killed. Then, you can avoid dephosphorylation, and avoid purifying the DNA, going directly from heat-killed digestions into ligation. The less you do to your DNA, the better.

[KhaleesiDany]

I have been doing some control ligations where i dephosphorylate half of the vector and other half not, i then ligated half of the dephosphorylated vector without insert though, and also the other half that was not dephosphorylated that too without insert..I was hoping to see alot of self ligated vector on the plates with the vector that was not dephosphorylated but instead i got non or 1.. i tried with 100 ng of vector even 500 ng of vector still nothing.. i tested my competent cells including my plates even the ligation kit used non of these seem to be a problem..we do however know that its the ligation of this paticular plasmid thats the problem... we going to try NEB ligase kit instead of thermo scientific.. do u have any ideas on what the problem could be or what i should do, what about using other ligases i have seen that T4 DNA ligase is not the only ligase there is T3? any comments on that.. thanks

[phage434]

Please check your competent cells by transforming with diluted plasmid DNA. It is very easy to fool oneself into believing the cells are good by transforming with high concentration DNA, which always works. You need high competence for this to work, and the only way to test for it is to try transforming very dilute plasmid DNA.

[KhaleesiDany]

Please check your competent cells by transforming with diluted plasmid DNA. It is very easy to fool oneself into believing the cells are good by transforming with high concentration DNA, which always works. You need high competence for this to work, and the only way to test for it is to try transforming very dilute plasmid DNA.

Thanks i will try this