just look at the concentration of antibiotics

Kanamycin concentration is ok 100mg/l. I don,t know whats a problem

There could be multiple problems. for ligation generally the reaction volume should not be more than 10ul. ligatons work perfectly undder smaller volumes owing to the fact lesser th space more is the probablility for DNA molecules to ligate. so ligation volume should have been a problem here...ligation perhaps did not work. did u set up a control ligtion to check ligase? secondly since the concentration of DNA is only 100ng, u shud consider using utr competent cells rather than normal competent cells. also the volume of ligation mix added to vol of competent cell is nearly half the volume of competent cells. so transformation efficiency would have been reduced too.  dd u set up control for transformation too with similar conc of DNA?

Hello,

We have all been in situations like this before. The first thing is the low concentration of your digested DNA fragments. I used to use one kit that have horrible results ~ 5 ng/uL and it made cloning difficult. Which extraction kit are you using to clean up your digestion reaction? Also are you doing single or double digestions? Sequential digestion and purification gives higher yields, but often a double digestion is sufficient to obtain the desired clone. Third, the ligation reaction can also introduce some complications. Depending on the vector, it is recommended to try a 3:1 and 6:1 ratio of insert to digested vector. It doesn't take much, I normally use ~ 100 ng vector and ~ 300 ng DNA insert in a 20 uL reaction volume. Try to increase the quality and quantity of your purified digests by incubation in a water bath for 10 minutes at 50C prior to eluting your DNA. If all of this fails and you are using a blunt end endonuclease or the cleavage of your site is low, you might design a sticky end set of enzymes or a more suitable restriction enzyme pair. Good luck, I'm sure you'll get it to work eventually!

Kind Regards,

Alex

thank you all. @ Thomas I use Axygen kit and did single digestion with SpeI O/N and i use total 20  ul volume of ligation as follow

T4 ligase 1 ul

10x buffer 1 ul

insert+ MYC  9 ul (9 ng/ul)

vector 9 ul  (2 ng/ul)

and grow on kanamycin plate 

For 20 ul ligation solution i need how much of T4 Ligase, Buffer, Insert (9ng/ul) & vector (2 ng/ul)??how much of this 20 ul ligation solution transformed into how much E. coli cells?

I would use a 30 uL reaction with

10 uL insert 90 ng

15 uL vector 30 ng

3 uL 10X buffer

2 uL T4 ligase

incubate for 1 hour at room temp (25C)

add ligase reaction directly to chilled competent cells

use 15 uL ligation mixture to a 50 uL aliquot of cells on ice

let stand for 10 min on ice then heat shock at 42 C for 90 seconds

return to ice for 5 minutes then add to 450 ul of LB or super broth and incubate while shaking at 37C for 1 hour

centrufuge cells for 5 minutes at 8,000 RPM, remove 400 uL of supernatant,

resuspend in remaining 100 uL and plate on Kan 50 and grow ON at 37 C

Hope that works and good luck!

Suggest me method according to my situation please. Thanks alot i will follow these steps. But i think concentration of DNA is tooo low in my case so it make hurdle so please tell me quantity of each ingredient which i use.

The volumes are given for your situatuon

10 uL DNA insert

15 uL plasmid

3 uL buffer

2 uL T4 Ligase

30 uL total volume

also try a control plasmid to verify the competence of your cells  

if it still doesn't work, try to increase DNA yield by starting digestion with more DNA or increasing the efficiency of your extraction  

Besides the previous interesting suggestions, I wold like to remind about VBNC: several microorganisms are viable but theu will not grow to form colonies.  It may be the situation.

Did you check the viability of your competent cells??

Yeah competent cells are ok no problem. I think low DNA concen is problem

If you have access, electroporation can give you better results.  But you would need electrocompetent cells.

Restriction:

The first thing you have to check is, if you can inactivate your restriction enzymes without cleaning it up again e.g. towards heat (if so you can just calculate back from the DNA which is actually in your digestion volume). If you have enzymes which can't be inactivated by heat just make more then one sample. For example if you normally make a 20 μl digestion with restriction enzymes A and B just make 3 to 4 of them to gain more DNA which you can clean up together over a column or else. 

Ligation:

For ligation it is important to have the right ratio! between plasmid and insert ( do you use plasmids at all???) 

Example: your plasmid has a size of 3000 bp and your insert 1500 bp. You want to have preferably 1:3 ratio between plasmid and insert ( it works best) 

Insert size / vector size (1500/3000) * 3(1:3) * ng concentration Vector in ligation (e.g.120)= insert-concentration in ng what you should use for ligation (180) 

I always made 20 μl volume based ligations

If you ligate over night @ 4 °C or for 1h @ rt doesn't matter or is highly philosophical...in my opinion

Good luck

Hi bushra

Usually I use 

6.5ul to 7ul insert for low concentration DNA

2-1.5ul plasmid

1ul T4 ligase buffer 10x

0.5ul T4 ligase (thermo scientific)

Incubate at 22 degree either in pcr machine or homemade water bath for 2 hours to overnight and then transform whole 10ul into 100ul of DH5a competent cells.

Hi Bushra, I am also doing cloning now a days and luckily I am getting excellent results  with Insert concentration of 10-15ng/ul after gel purification. I am also using TOP 10 competent cells. I am giving some suggestions along with some gel images for your help.

1. The most important thing in case you are not getting your results is to be calm and relaxed when repeating the experiment. Frustration and rushing into things while doing experiment does not help at all. Everything goes wrong.

2. In case you are using a PCR amplified product (as Insert) for enzyme digestion, make sure the restriction site is not close to the ends and should have sufficient length (bases before restriction site) for RE to work. (I successfully cloned both PCR amplified product and a non amplified one from vector)

3. For setting up ligation, calculate how much of insert and vector volume you need. For this, I use NEBiocalculator. I am giving you my today`s ligation result. I double digested my Vector and Insert (One of my enzymes produces blunt while the other produces sticky ends.) , then gel purified. Concentration of vector and insert is 41 and 29ng/ul respectively. My Vector is 4kb while my Insert is 1.2kb. When I calculate MASS TO ENDS using NEBiocalculator, 1ul of my vector is equal to ~33fmol while 1ul of insert is equal to ~80fmol.  Now for ligation 1 of ratio 1:3,  I took 1 ul vector and 1.2 ul Insert. For second ligation I kept the same ratio but doubled the concentration: Vector 2ul, Insert 2.4ul (I am not trying any other ratio here bcz I already know from my previous experiments that ratio 1:3 works well for my experiments). So its 2 ligation reactions Lig1 and Lig 2. 

Ligation rxn=20ul, incubation time 60min, Room Temp (I used Invitrogen T4 DNA ligase 1ul per rxn)

Run 5 ul of ligation on 1% agarose gel along with digested Insert and digested vector.

You can see the results in the gel image I attached. Lane 1 is 1kb ladder, 2. digested Vector 3. digested Insert 4. Ligation1 5. Ligation 2

I always run ligation rxn on gel to confirm if my ligation is working or not. It saves a lot of time and money.

4. Once you get the idea that your ligation is working, now proceed to transformation step. In my case I use 5ul of the ligation rxn to transform 50 ul TOP10 E.coli. Fresh stock of competent cells is recommended

In my experience, TOP 10 are more easy to transform than DH5 Alpha strain.

I have also successfully cloned 1.5 kb Insert into a 5kb Vector in a similar manner.

And in case your Insert or Vector concentration is too low after gel purification, just reduce the volume of distilled water u add at the end of purification step to dissolve DNA. the concentration will increase. For example I use 10-15 ul DW to dissolve my DNA at last step of purification.

Hope this helps. Salam

Thanks alot, but you directly run ligation on gel without doing pcr & how we know from gel that our ligation is better?

Yes I run ligation directly on gel without pcr as I have done it in the image provided in my previous comment. How you know from gel if ligation is successful,,, just have a look at the gel image again. Lane 2 is digested (means linear) vector, Lane 3 digested (means linear) Insert. Lane 4 and 5 are ligation rxn run directly on gel after 1 hour incubation at room temperature. compare the sizes now. If ligation is working you should get the desired size band on the gel. In lane 4 and 5 you can see multiple bands. They normally represent Linear, relaxed circular and supercoiled conformations. In some cases other bands can also be visible representing supercoiled denatured and nicked open conformations. In the above image my vector is ~4kb and insert is ~1.25 kb and I can easily see a band of app 5.2 kb. 

I have already transformed the above ligation in TOP10 E.coli and have successfully grown colonies from which I isolated plasmid and confirmed through sequencing.

@Abdullah Abdullah, i extract DNA from gel after restriction digestion of over expression vector & gene of interest with SpeI, but I get very very low concen about 0.9ng/ul in case of over exp vector & 5.6 ng/ul in case of insert. I did ligation of total 20ul reaction in which i add 8 ul insert, 4 ul vector, 1 ul T4ligase, 1ul buffer, and remaining dd water and keep it overnight 4 degree. In morning i did transformation and next day i find no colonies on plate.When i run gel i get no band in case of ligation.  What is reason?? why ligation is not success ful. thanks

Hi Bushra, if you want me to help you then please provide me some more details about your vector (vector name, size and company), your Insert sequence (just sequence). I also need to know how you setup restriction digestion. You can contact me through my email address ([email protected]). Please also mention whether you amplify your gene of interest before restriction digestion or not. I shall try to help you as much as I can. You also need to make sure your enzyme is working fine. have you tried to check that?

@Abdullah Abdullah, pCUBI1390 is expression vector i use. I did single restriction digestion of both vector and gene with SpeI by using total 30 ul volume contain 1 ul SpeI, 5 ul buffer 4, 0.5 ul BSA, 2 ul plasmid (insert) (120ng/ul) and expression vector 2 ul (66ng/ul). My gene of interest is in pEASY T vector, i dont amplify it. Thanks alot

Hi Bushra,

1. So your insert is in p Easy T (pGEM Easy T vector 3kb). And you are trying to clone it into pCUBI1390 vector. Right? pCUBI1390 (10kb) contains a single SpeI restriction site. Similarly your pEasy T vector also contains an SpeI restriction site at position 95 (I got this information from internet). Please let me know if the above information is not correct. I could not open your insert seq file otherwise I would have checked if it contains any SpeI site. Please check for that. If there is no SpeI site in your insert sequence then you r trying to clone one whole vector into another 10kb vector. Please let me now if your insert sequence contains SpeI site and if yes then how many.

2. Have you checked if your SpeI is working fine. (run undigested and digested  pCUBI1390 plasmid on gel, you ll get the idea, undigested plasmid should show multiple bands while digested will show only one discrete band)

3. When you run digested Insert (which is in pEasy T vector) on gel, how many bands do you get. 1 or 2?

4. Problem with your restriction digestion: You are using only 240 and 130 ng of total plasmid DNA for restriction digestion while recommended concentration is 1ug. I always use 1 ug plasmid DNA. If you use low concentration in restriction then you are going to get very low concentration after gel purification.

5. Did you check if your Ligase enzyme is working or not?

Best Regards

If your Insert is in pEASY-T1 vector (not pGEM EasyT vecotr), then it also contains a single SpeI site. Please provide complete details.

Yes Sir you are right, yeah pEASY T vector also contain SpeI site. I did not amplify my gene of interest from pEASY , directly use the right colony. When ever i do restriction digestion i get clear bands of pEASY vector but very very light bands of my gene of interest and after dna extraction concentration is always very low. My gene of interest itself dont contain speI, but i add speI sites on its primers. SpeI & ligase work. pEASY give me single band after restriction digestion, & my gene of interest also give single band.

You said you directly used the right colony. Please tell me how did you confirm that the colony you picked actually contained your gene of interest. if you did sequencing for that, were SpeI sites present in the sequence?

I cannot understand your sentence "pEASY give me single band after restriction digestion, & my gene of interest also give single band". What I wanted to ask is that your gene of interest is already cloned in pEASY T. When you digest your this vector with SpeI, then how many band you get on gel. you should get 2 band, one of your insert and the other of your remaining pEASY T plasmid. Do you get your insert correct size on gel? If you are getting single band after digesting your pEASY (containing your gene of interest)with SpeI then either SpeI site is missing or gene of interest is not there.  Let me know about this. Then I can suggest you accordingly.

In case you are sure that your pEASY T contains your gene of interest and after digestion you get band for your gene of interest, then the main problem is the very low concentration of Insert and Expression vector in ligation reaction. What is your insert size? I already told you that you are using too low concentration in restriction digestion which after gel purification will always be very low. 

1. You have big problem with ligation. You said for ligation, you used 4 ul of 0.9ng/ul in case of over exp vector (10.9kb in size) & 8 ul of 5.6 ng/ul in case of insert (size??). Now did u made calculation for this. 4 ul of 0.9 ng/ul of your 10 kb vector is equal to 1.069fmol. While recommended moles in ligation rxn for vector is about 30 fmol. The ligation is never going to work in your case. You keep repeating and it is never going to work at all. Similarly recommended moles for Insert is 90 fmol. Make calculations and then proceed.

You should know that the ligation ratio of 1:3 (vector to insert) is not based on DNA concentration (ng/ul) only. It is the vector to insert molar ratio as well as vector ends to Insert ends ratio (ratio of number of moles). This molar concentration changes with size/length of DNA fragment. Check your DNA ligase document.

Use NEBIOcalcultaor as I already explained previously.

2. You should consider that the size of your expression vector is relatively big (10.9kb) and it will not be easy to ligate insert when you use such low concentrations.

2. You said you use 2 ul of 66ng of expression vector in Restriction digestion. This is again a problem. If you use such low concentration your are going to get not more than 5-10ng of your expression vector after gel purification or even lower than this. Same is the case for your insert concentration. You need higher concentrations. Grow your E.coli colonies containing your expression vector and also grow E.coli colonies transformed with your insert containing pEASY T vectors in LB broth and isolate plasmid. Use only 10-15 ul D.W or buffer at last step of plasmid isolation to get higher concentration

3. For getting higher insert concentration there is also an alternative method. Use M13 primers to amplify your gene of interest. then run 1 ul on gel to confirm PCR is successful. Once pcr is confirmed then transfer remaining PCR rxn into spin column of gel purification kit. Add 5 volumes of gel purification buffer (normally yellow in color) directly into pcr rxn and mix well. Then centrifuge and continue with rest of purification steps. You will get clean pcr now. Use 10 ul distilled water at last step of purification to dissolve DNA. Measure DNA concentration. Use appropriate amount for restriction digestion.

Hope my suggestions work for you. Best of luck

Insert size is 1 kb. If i follow your last option to amplify my gene of interest, is there any chance of mutation? Please check file, what it mean, how much insert dna and vector i take in ligation in case of microliter.

In my case I never experienced any kind of mutation when amplifying my insert. Later you can confirm through sequencing.

For ligation, you are not getting my point. The picture is from ligation calculator, it only tells you how much insert you should take relative to your vector concentration whcih was 0.9ng. Your Insert was 5.6 ng so make calculations . But this is not going to work at all in my opinion. Your Vector concentration is too low. If you increase this concentration to 50ng in the software, the same software will give you different result.  Ligation calculator gives you calculations only. It is not going to tell you whether 0.9ng or 50ng or whatever concentration it is is enough for ligation to work or not. . 

But I am giving you this suggestion again that you need to understand the concept of ligation. Its not only about molar ratio. Its about free 3` and5` ends available for ligation. I will expllain very shortly. If your vector is 10kb in size and concentration is 10ng/ul, it will contain less 3` and 5`  free ends for ligation as compared to a 3 kb vector whose concentration is same (10ng/ul). Use ds MASS TO ENDS calculator on NEBiocalculator to calculate ends. I use Invitrigen T4 ligase. Plz check the attached t4 ligase document. You can see vector ends and insert ends mentioned there.

thanks alot Sir. 

Hi Abdullah Abdullah , I am doing self-ligation now and also have some problems with the ligation efficiency, my DNA is 4.02kb and I usually use 50ng of DNA in a 20uL of ligation reaction. After reading your replies above, I calculated the moles of my DNA ends, it is 40.26 fmol. Is it enough for a self-ligation?