Hi Suraj,

I want to know something about your experiment

1. Did you check your digestion is OK or not? Have you done single digestion or double digestion? How long your double digestion and under what conditions? Are you purifying your cut vector from gel? Or did you treat your vector to restrict self ligation?

2.How long the distance between your RE sites in vector? If they are too close(less than 6 bp) to each other, then the second enzyme will not cut. You should keep in mind there are some key points to select correct RE otherwise it will give you lots of headache.

3. What is your Insert: Vector ratio? It's very important in case of ligation.

4. How long is your target? If it is too long then you can go for infusion technique.

5. What condition you keep for ligation? I mean the time & temperature of incubation.

6.Transformation condition ( If you get colony then I think your competent cell & transformation condition is OK)

Here I want to share with you some of my points

1. Target PCR product with compatible restriction sites of vector/plasmid ( Your selected restriction site should not be present in your target sequence otherwise it will cut the target inappropriately)

2.You must have an idea that your restriction enzymes form blunt end or cohesive end. If it is form cohesive end then you can easily use for ligation but if it is produce blunt end then you have to treat. You can set the treatment according to the protocol you got from company of your ligation enzymes.

3. After digestion you can confirm the digestion is OK or not by gel run.

4. In case of vector you can treat digested vector with CIAP enzymes to restrict the self ligation of vector that is may be the cause that you got some false colony.

5. You should clean the digested target & vector to remove unwanted fragments

6. If you confirm that your digestion & purification is OK then set the ligation system. Ligation system & condition based on the company ( Thermo scientific, NEB or TAKARA) protocol who provide you the ligation enzymes & the type of enzymes( T4 ligase or other) you used.

7. For ligation 10-20µl system is OK. I have found the ratio of Insert : Vector; 3:1 to 5:1 is working very nice & 1µl of your ligase is enough for good result.

8. I found the incubation condition is depend on ligase enzymes you use. But in case of most ligase I found overnight incubation at 4°C or 16°C showed good result, 30 min-1hrs at 22°C or 2-4hrs at RT also work .

9. If you confirm about your competent cell & have confidence on transformation protocol then hopefully you will got your correct ligation.

Hope it will work for you.

Wish you success in your research.

Due to frequent thawing ATP in your buffer might have degraded, so add ATP externally or take a fresh buffer n try, it may work

Hi Suraj,

I want to know something about your experiment

1. Did you check your digestion is OK or not? Have you done single digestion or double digestion? How long your double digestion and under what conditions? Are you purifying your cut vector from gel? Or did you treat your vector to restrict self ligation?

2.How long the distance between your RE sites in vector? If they are too close(less than 6 bp) to each other, then the second enzyme will not cut. You should keep in mind there are some key points to select correct RE otherwise it will give you lots of headache.

3. What is your Insert: Vector ratio? It's very important in case of ligation.

4. How long is your target? If it is too long then you can go for infusion technique.

5. What condition you keep for ligation? I mean the time & temperature of incubation.

6.Transformation condition ( If you get colony then I think your competent cell & transformation condition is OK)

Here I want to share with you some of my points

1. Target PCR product with compatible restriction sites of vector/plasmid ( Your selected restriction site should not be present in your target sequence otherwise it will cut the target inappropriately)

2.You must have an idea that your restriction enzymes form blunt end or cohesive end. If it is form cohesive end then you can easily use for ligation but if it is produce blunt end then you have to treat. You can set the treatment according to the protocol you got from company of your ligation enzymes.

3. After digestion you can confirm the digestion is OK or not by gel run.

4. In case of vector you can treat digested vector with CIAP enzymes to restrict the self ligation of vector that is may be the cause that you got some false colony.

5. You should clean the digested target & vector to remove unwanted fragments

6. If you confirm that your digestion & purification is OK then set the ligation system. Ligation system & condition based on the company ( Thermo scientific, NEB or TAKARA) protocol who provide you the ligation enzymes & the type of enzymes( T4 ligase or other) you used.

7. For ligation 10-20µl system is OK. I have found the ratio of Insert : Vector; 3:1 to 5:1 is working very nice & 1µl of your ligase is enough for good result.

8. I found the incubation condition is depend on ligase enzymes you use. But in case of most ligase I found overnight incubation at 4°C or 16°C showed good result, 30 min-1hrs at 22°C or 2-4hrs at RT also work .

9. If you confirm about your competent cell & have confidence on transformation protocol then hopefully you will got your correct ligation.

Hope it will work for you.

Wish you success in your research.

A good comprehensive answer above. IN short, have you used a double digest? One of the problems with this process is that you don't have any checks in place.

The best thing to do is to SUB-CLONE into a TA cloning vector e.g. pGEMT!!!!

1) You can digest out the insert from this vector and be sure both ends are digested corretly.

2)You will have lot of the product (always makes thing easier)

3) Using a double digest will ensure you do not get self-ligation of the pET vector.

Hope this helps. The sub-cloning step is the key here.

You take out your ligation buffer and thaw it, vortex it, check the smell of DTT. if there is no smell that means your buffer is degraded and ATP as well. so try the ligation reaction with new buffer and after thawing vortex the buffer nicely and then immediately use it for reaction..

you can try ligation with PEG also, sometimes it works..and keep in 4 degree centigrade for 16-20 hrs that gives you better results.You can also try with the vector: insert ratio of 1:6 or 1:8..

Good luck.

Both of your suggestions are highly appreciated.

@ravi: sir I have checked my buffer using new buffer also but it didn't work.

@shajia: I have checked the digestion and it was OK. I am using all the enzymes and buffers from NEB. I have kept my ligation reaction of ratio 1:3, 1:4 and 1:5 at 16°C for 16 hrs, 22°C for 3-4 hrs and RT for 2 hrs (as literature suggested), but none of the conditions work. The DNA fragment i want to clone is 840bp. I can assure you that my comp cells are working well after transformation.

I want to know if there is any way to check my ligase enzyme whether it is working or not??

It is decades ago that I did extensive cloning, but maybe you try several different vector:insert ratios (as others suggested). You may try even 1:10 to 10:1. Also: you may start the ligation at cold temperature 4-12 degrees Celsius (for example over night/day, 24 hours, then add again ligase, another night, and finally add another minimal volume of maybe one microliter ligase and heat for one hour to 30 degrees Celsius... If none of the other suggestions worked, you may try this. Without garanty.

You have written that you are getting colonies but they are not of your interest. That may be due to self ligation of your pET.

So, try to keep following things in your mind:

First be pretty sure about double digestion of your pET vector by transforming of your double digested vector as a control. In result you should not get any colony.

2. When you confirmed that your pET is double digested then you go ahead with your protocol for ligation. I think it works.

May be you are getting ligated colony but due to background of false colonies you are missing right ones.

Hi

Before transformation, I recommend to check this steps:

PCR product should have seating site for digestion enzyme. If the restriction sites are placed at the PCR products, with no additional base, the digestion efficiency of linear fragment is very low (of course not impossible).

Inactive the restriction enzymes (by heat traetment,...) after digestion.

The ratio of insert and vector should be determined with regard to pmol ends.

You may have a ligation control reaction that includes vector + buffer + enzyme (with no insert) to check uncut vector and self ligation rates.

To be assured of ligation, you may use 1 microliter of ligation product in a PCR reaction with vector primers. You'd better to run PCR for 35 cycles.

If everything is OK, you may notice to Insert. Any proteins could have toxic effect on host, if highly produced. To overcome this, You'd better to use SOC culture medium in all the steps of bacterial transformation. After transformation, incubate at 25 degrees, after about 3 days, small colonies will visible (It is necessary to transform the ligation control reaction).

God speed

Hi, I think that all the suggestions above are really good.

I only want to say that to know if your ligase work well you have to use this enzyme in another cloning experiment, if you are afraid can you ask some coworkers to give you another enzyme?

Control your antibiotic, probably you used fresh LB agar dish, check that your antibiotics are good.

From my personal experience:

I agree with Ian and Bahar, when I try to digest my PCR product and cloned directly in my vector I did not have colonies, sometimes the restriction enzymes can't digest well at the end of your PCR products (even if the PCR is good, even if you attached your restriction enzymes sequences), and when you check the fragment on gel you can not tell if the digestion work well or not!

For this reason is better to clone your product in a TOPO cloning ( or pGEMT, but I prefer the TOPO!) because it is a plasmid designed to ligate, without problem, a PCR product (it is less expensive if you buy the plasmid without the competent cells, and failed trasformation it is also expensive!).

I think that you perform all the thing correctly and you follow the manual instruction, so good luck and don't give up!

To make sure ligation successed, I often do PCR with 2 primers located on plasmid backbone using ligated solution as template (1ul). And then pcr product size is aboyut sum of your insert and plasmid backbone. If your insert quite large, you can use one primer on plasmid backbone and another designed on insert for shortening pcr product, more easier to confirm. Then, confident to move to next step, transform to competent cell.

Hi Suraj, you can check your ligase work or not by re-ligating a single digested plasmid. Digest your plasmid with single RE, clean it & treat it with CIAP for 1hr at 37°C. Clean CIAP treated plasmid (keep some for transformation as a control) & set your ligation reaction. Transform ligated plasmid & at the same time transform CIAP treated plasmid as a control. You should not get any colony with digested plasmid but get colony with ligated plasmid if your ligase work.

* CIAP treatment is not mandatory for your test but you can try for satisfaction.

You can also run gel with your ligated & digested plasmid. If ligation happened you will get larger single band then digested plasmid.

You can also do PCR using your plasmid/digested plasmid & ligated plasmid as a template as Bahar Zare suggested you.

*Please clean the ligated plasmid when you want to use as a template.

I am little concern about your RE enzymes. How many bases you added before RE sequence as a protective base? Did you check the compatible cohesive end of your selected RE? How many colonies you selected? You did digestion check or sequencing or both for confirmation?

Hope you will get rid soon of it.

For a lot of complicated cloning, I ended up with only blunt-end cloning.

You feel that your PCR worked fine. It may not be so. You need to optimize the denaturing and annealing cycles properly besides other factors like the buffer, primer concentration etc.