Ok i will dephodphorylate vector. Is the above concentration is ok for ligation? I tried many times but concen always low after restriction digestion.

Did you have insert in a background and are you are releasing the fragement by single restriction digestion with Spe1. If you want to do colony PCR to check if ligation has occured or not, then both forward and reverse primers should have Spe1. 

First digest the vector with Spe1 and then do alkaline phosphatase treatment to prevent self ligation. You can set up a reaction by adding the restriction enzyme Spe1 and Alkaline Phosphatase to the Vector. Most of the restriction enzymes and Alkaline phosphatase have activity in the single buffer assuming both Spe1 and Alkaline Phosphatase are from same company. Once the reaction time is over, deactivate the alkaline phosphatase by heating at 65C for 15 minutes.

Regarding insert,first check on the gel after restriction digestion if the fragment is getting released or not  by adding the digested product in the gel. After confirming the release of the insert after restriction digestion, carry out gel extraction by cutting the insert fragment from the gel. Elute the insert in minimum quantity of elution buffer such that u get insert in higher concentration. Since there is great loss of DNA during gel extractionkit , set up 4-5 reactions for the release of gene of insert.. 

Set up the ligation reactiion with restriction digested, dephosphorylated vector with insert in different ratios of 1 :1 - 1:5.  Ligation carried out at 16C. Transform the highly efficient competent cells of DH5 with ligation mixture and plate on the medium. Once colonies appear, carryout  Colony PCR with forward and reverse primers for the gene of insert.

Use restriction enzymes from standard company such as ThermoScientific, Fermentas, /NEB. 

There are many online calculators for setting up a ligation reaction.

http://2011.igem.org/Team:UT_Dallas/ligation

http://www.insilico.uni-duesseldorf.de/Lig_Input.html

Above all, Cloning is very tricky. It wil take quite a good period of time to get transformants, Keep doing with patience until you get success. 

I just had the same experience and same pattern of work in which you are facing problem. Initially, i was trying to digest the vector of 15 plus Kb with Hind111 and linearising it. After confirming the digestion, i used to purify it with PCR purification kit and then use the eluted product for dephosphorylation with Alkaline Phosphatase. Again i had to purify it and then elute it. As a result there was huge loss of vector due to clean up two times resulting in a poor yield in the final product. Hence, i worked on that and found that when we use same company Restriction enzyme and Alkaline Phosphatase , there is no needup to clean up the product and no need to perform the digestion and dephosphorylation separately. We can initially set up a restricton enzyme digestion say with Fast digest Restriction enzyme of Thermoscientific. After digestion is completed, inactivate the enzyme at the mentioned temperature (Say its 80 C for 20 minutes in case of Hind111), Check what is the deactivating the temp for Spe1, Then add the alkaline phosphatase tot he same product without purifying the digested product . Alkaline Phosphatase and Restriction enzyme have activity in the same Fast Digest Restriction Buffer. After phosphatase activity for 30 minutes, inactivate the phosphatase activity by heating at 65 C for 15 minutes. After this no need of purifying it. 

Second, you release the insert from the background by single digestion and run on the gel. Gel elute the released fragment. For this you should setup 4-5 reactions. 

Peform ligation of eluted insert and the digested dephosphorylated vector and incubate at 16 C overnight. 

Use the ligation mixture to transform DH5 Alpha and plate them. Check for insert fragment by doing Colony PCR with primers for insert. This process saves lot of chemicals , vector. I struggled a lot for 35 days . But i got success when i followed the above mentioned procedure

You should better to mention the name of vector and insertion and their sizes, then the suggestions will be better to follow. If the colonies are grown but you cant get the DNA after isolation, your antibiotic(s) that you used in the medium might be impure or not working. So you should use new antibiotics of the same. Self ligated colonies will be separated as blue colonies when you use Xgal and IPTG or others as necessary in the medium (white colonies are the vectors with insertions when you want to insert in the Lac region of pBluescript, pUC etc). Its very easy to separate the candidates from self-ligated ones. 

Yes. it is important to mention the vector and insert and their sizes. May be not to mention the work before publishing but vector name , size can be mentioned with out naming the insert. Insert size may be mentioned. When we suggest , we feel that assuming conditions are optimal or all the reagents are prepared properly and they are working at their optimal levels. Amount of vector and insert to be taken for  setting up of  ligation also depends on the concentration, size of vector, insert.

I use pCUBI 1390 over expression vector (10860 bp) and insert size is (1000bp). In this case i dont use X gal and IPTG, so no difference.I change antibiotic many times, use fresh one. I dont know whats problem

Get an idea of all the suggestions by the followers of your question and make an attempt to do ligation. It will work. Since vector is around 11Kb and insert is 1 Kb, its not significantly difference to check on the gel. Only thing you can do is after getting the colonies, isolate the plasmid after getting colonies and do restriction digestion to release the fragment. Continue to make attempts and you will get success.

Can i use Alkaline phosphatase after restriction digestion extracted DNA OR must use during restriction digestion process? I order NEB and protocol is as follow.

Digest 1–5 μg of plasmid DNA in a 20 μl reaction as follows:

DNA ≥ 1 µl

Restriction Enzyme Buffer (10X) 2 µl

Restriction Endonuclease 1 µl

H2O, purified to 20 µl

Note: Scale larger reaction volumes proportionally.

Incubate at 37°C for 60 minutes or follow manufacturer’s recommendations.

Add 1 unit of rSAP for every 1 pmol of DNA ends (about 1 μg of a 3 kb plasmid) and incubate at 37°C for 30–60 minutes.

Stop reaction by heat-inactivation of rSAP and restriction enzyme (follow manufacturer's recommendations).

Note: If restriction enzyme cannot be heat-inactivated, DNA purification is required before ligation.

What is means, i add how much rSAP in 50 ul reaction and can i keep it over night with SpeI at 37 degree? SpeI deactivation is 80 degree for 20 mins so how deactivate rSAP? Please help me to simply protocol that i easily understand . thanks

You can use either of the process with simultaneous restriction digestion and dephosphorylation or first complete the restriction digestion and then inactivate the Spe1 and dephosphorylate the vector. If we think logically, first we complete the restriction digestion so that plasmid gets linearised . Then the 5' and 3' terminal phosphates are available for alkaline phosphatase to act and dephosphorylate. Then we can inactivate the Alkaline Phosphatase by heating at 65 C for 15-20 minutes. You use this mixture to do ligation with the insert by T4 DNA Ligase. Since we are not purifying the reaction from Buffer, Restriction Enzyme, Alkaline Phosphatase we cannot calculate the exact concentration of the digested dephosphorylated vector to ligate withthe insert. But still we can add gel eluted insert and ligate it  . Then transform teh ligation mixture into DH5 Alpha. This seems to be easy. On the other hand we need to setup 8-10 reactions for digestign the vector and linearising it. Then gel elute the digested product where we lose good amount and then set up the alkaline phosphatase treatmetn and again do PCR clean up/Gel elute which again make you lose digested dephosphorylated plasmid. You will end up with little amount of vector making you difficult to set up a ligation reaction with insert. So, i suggest you use gel elution only for releasing the insert fragment from background vector in which the insert is cloned. 

Hope this works and let me know if you have any other queries or assitance.

Good Luck. 

Thanks alot Sir. I already digest vector & insert with SpeI, so can I add Alkaline phosphatase in purified DNA, and how much microliter i  add? I add some buffer with it or no need? and keep it for 30 mins 37 degree?then deactivate it 65 degree for 20 min?

Is the Spe1 and Alkaline Phosphatase you have of same company? Is the buffer you are using for restriction digestion has activity for Alkaline phosphate also ? Check online for the same. Inactivate the Spe1 and add alkaline phosphatase . I unit or 1 microliter sufficient to digest one microgram of DNA. Based on the total amount of plasmid DNA you have taken, add the volume of alkaline phosphatase accordingly. Are you doing normal digestion of Fast Digest FD Enzymes?

i am doing normal digestion, both are from NEB. But I am not sure wether they work in same buffer or not, I will check. thanks

Both Spe1 and Alkaline Phosphatase have functional activity in Cutsmart Buffer. You can carryout the reaction with that. Could you please let me know what buffer you are currently using. I will be glad i can assist you further if you can give me further details. If it is normal buffer you need to incubate for more time than Fast Digest buffer. If the incubation time exceeds too much some restriction enzymes have star activityresulting in digestion at random sites other than the restriction sites. You need to take care of that also. With Fast Digest enzymes you can carryout reaction in shorter span of times .Also, these High Fidelity restriction enzymes have dramatically reduced star activity which is a pretty good feature.  For the time being, you can carryout the reaction with the enzymes you have . But dont incubate at extended intervals than recommended. Little bit fluctuation is OK.

Are you sure your Restriction Enzyme is fine and not expired. After setting up the digestion with Spe1 for release of insert from the backbone vector, your gene of interest is getting released?

I am using cut smart for SpeI. Yeah SpeI is fine, no problem , I change it recently. Yeah it give me ood results but insert is sooooo light because it is in pEASY vector.

I ever failed to ligate too , then I try to ligate first using Mighty clone then after I get the PCR product from the Mightys' plasmid, I ligate with the target vector without dephosphorylate. I'm usually incubate 37 degree for 30 min then 70 degree 5 min for inactivated the enzym. 

hope can help

This is colony PCR of Shrimp Alkaline Phosphatase vector, I get right bands and send samples for sequencing. Why some bands are too big and some are very thin?

Bushra Khattak. It depends on the inoculum that is coming with your microtips . More template more amplification till optimum levels . Less template less amplification. Too much of bacterial colony you get a smear with out any amplification. Generally when you do colony PCR, you streak the bacterial colony on  the plate and immerse the microtip in the PCR mix