Few precautions that i am aware of as we have done it with the PCR products of up to 7 Kb;

1. Do not use more than 30 cycles for amplifying your PCR product, if you need more amount run it in 2-3 tubes but do not over cycle.

2. Use good proof reading enzyme like Phusion polymerase.

3. Do gel extraction with kits like QiaQuick gel extraction kit (Qiagen) so as to get more amount of product after digestion and gel extraction.

4. Run gel in TAE buffer

5. Finally, use good ligase and competent cells.

No specific reason why it should not work, did it dozens of times and it mostly worked on the 1st attempt.

* Try to use high quality, established restriction enzymes

* Make sure you have enough PCR product

* If you cut close to the end of your PCR product, make sure there a some extra bases in order for the enzymes to work properly

* Check your digested plasmid before you waste your precious digested PCR product

Good luck!

Hi there,

The most important is polymerase fidelity so go for Phusion!

Thank you very much Maximilian Peters and Kamal kumar sir for your valuable suggestions, Here i have tried with TAKARA Ex Taq DNA Polymerase for amplification of 3 Kb insert, and i have added 3 extra overhang nucleotides with addition of added enzyme restriction sites (EcoRI and XbaI) in primers, repeated this experiment 5-6 times always got only self ligated clones. Is it due to improper digestion of vector (used Fermentas Fast Digest enzymes for 3, 6, 9 and 12 hrs) or it is due to improper digestion of insert due to less number of added extra overhang nucleotides. Should i add more than 3 or it is sufficient for enzymatic digestion. Please suggest...

* Can't comment on the polymerase, never worked with it, but if you get enough PCR product it should be fine

* For the primers I usually add 6 extra bases before the restriction site. Three looks border line for XBA, according to:

https://www.neb.com/~/media/NebUs/Files/Chart%20image/cleavage_olignucleotides_old.pdf

* For the vector: Run an empty control without your PCR product, that way you know if your vector is only partially digested or your PCR product is the problematic part. My suggestion: digest with your enzymes of choice, dephosphorylate, purify on a gel, try to run it as long as possible, (yes, it takes more time, you lose a lot of DNA but any partially digested plasmid will be removed and will not contaminate your transformation).

Dear Maxmilian Peters sir, I am getting enough pcr product after 30 cycle and after purification i am getting good quantity too, Sir, during digestion of vector in any case it will linearized only, ( only 12-18 bases from Multiple cloning sites will be released after double digestion) if both the enzyme works fine or single enzyme works and it is quiet difficult to decide whether our vector digested properly from both the ends or not. Sir, please give me some suggestions to overcome from this problem.

If you were using both enzymes in the same buffer, cut the plasmid with each enzyme seperately and run them on a gel. If you see more than one band, you are in trouble and should get new enzymes.

If you digested sequentially, change the order of the enzymes and purify on a column in between.

if both the enzyme works fine or single enzyme works and it is quiet difficult to decide whether our vector digested properly from both the ends or not.

You are totally right, the gel purification just gets ride of minute amounts of undigested plasmid which you won't see on a gel but will screw up your transformation.

Did you dephosphorylate your plasmid? This step usually greatly reduces background reactions.

Dear Maxmilian Peters sir, I haven't tried the dephosphorylation of my digested vector yet in this case, i thought this will work in case of single enzyme digestion to prevent self ligation, but in case of fully double digested vector it should not ligate logically. Anyway sir, i will dephosphorylate my vector after double digestion this time, lets see what happens ? Sir, what should be the optimum duration for this treatment as because i have read in some literatures that prolong incubation also damages the ends of digested vectors, so optimum duration should be required.

Theoretically you are totally right but E. Coli under selection stress are masters of survival, even if you use mutant strains, they will do anything with the plasmid in order to survive.

For the dephosphorylation protocol, stick to the manufacturers instruction, incubation time depends on the specific phosphatase. For most reactions I was using FastAP from Fermentas which needs only 10 minutes to work. So double digest with restriction enzymes from NEB for 80 min, then 1 µl of FastAP for 10 min at 37°C and either straight on a gel or in the -20 freezer.

Good luck!

Please keep us posted, so others can learn from your experience :)

Dear Maxmilian Peters sir, Definitely I will post whatever i will get the results and troubleshoot all the incoming problems by discussing with you all and getting your valuable suggestions. 

Thank you very much

Yes. If you follow the instruction manual of Invitrogen but practically I tried it is bit difficult. You can design internal primers (nested PCR) and check its amplification.

all the best

I have not tried to do this myself, but keep in mind that restriction enzymes don't really like to cut near DNA ends. You may have to use quite a bit higher concentration of enzyme than is suggested by the supplier  based on cutting of "typical" sites. Also often best to cut with two enzymes sequentially with de-proteinization and cleanup in between. Also that way can use optimized buffer conditions for each enzyme, instead of forcing them to work in one all-purpose buffer. I wouldn't think you'd need to gel purify in between digestions, but you may want to gel purify the primary PCR product to get rid of small stuff that you don't see but is probably still in there (leftover primer, primer dimer, etc).

Thank you very much Mark Elliott Samuels and all,  everyone have given a very good suggestions, Here i am following same, I did sequential digestion of my insert pcr product (full length geminiviral genome 2.8 Kb) and vector with EcoRI and HindII using fermentas Fast digest enzymes, further dephosphorylate my desired vector too and finally set up the ligation and transformed into E. Coli competent cell, but again i did not observed any type of blue or white colonies over ligation plate. Now, it is very difficult to know that either my insert pcr product has not digested properly or vector.