Hi Shanice, 

If you were my student, this is how you would be doing it.

1. Clone your PCR product into an intermediate plasmid, such as pGemTEasy, using TA cloning

2. digest both plasmids with restriction enzymes in a double digest (providing they are compaible in the same buffer - sequenctial digests are only useful if they are not compatible)

3. gel purify both insert and vector

4. determine concentration of insert and vector, and use a molr ratio of insert 3:1 of 5:1 (maybe higher ratio if insert is big), with 50 ng of vector

5. ligate overnight at 16C

6. transform.

By doing the initial ligation into a random plasmid, you will find they restriction digestion efficiency to be much higher than directly cutting the PCR product. You can also sequence that product, and make sure it is correct before proceeding any further - you'd hate to get to the end only to realise it was wrong. By doing the gel extractions, you can make sure you get the right product, and it helps filter away non-specific products. By measuring the concentrations, you know EXACTLY what is going into each reaction. Ligating overnight will improve efficiency. Dont bother running the ligation mix on a gel - it wont tell you anything constructive.

I hope that helps somewhat. If you want some more specific feedback, it might be helpful to outline some of your actual protocols.

Cheers, 

Steve

As you told you got the clone consisted of your insert means ligation efficieny was not good. However, some of your insert ligated into vector. You can not see them on the gel electrophorsis due to low copy number. So continue your jobs. bye bye!!

Hello Shanice,

Here are my suggestions for a more efficient experiment.

1. try to do digestion of your PCR product and vector, one enzyme at a time. Make sure you start with at least 1 ug of DNA. Digest your PCR product and vector with one enzyme first. Then purify. Then digest with the second enzyme, and then purify the digested DNA.

2. I presume you are using the regular restriction enzyme since you are incubating the mix overnight. I suggest you use a FastDigest Enzyme by ThermoSci. You can do the digestion in just 30-60 minutes.

3. Make sure your ligation mix contains more vector than the insert. usually, I use a 1:5 ratio (vector to insert).

4. I usually do not run the gel for my ligation mix, since I am sure that my mix contains the right amount of DNA. I go for transformation right away.

I hope this helps.

Kris

Hi Shanice,

I don't see any obvious fault in your strategy, but what do you mean with "the clones I get did consist of my insert"?

The steps that may go wrong are essentially an incomplete (or too much) digestion or a failed ligation.

Anyway, there are several other strategies for such cloning. You can consider subcloning the amplified fragment to another vector first, grow it, check the sequence, then cut and isolate the insert from the gel. this way you can be sure that the fragment you take is doubly cut. Alternatively you can try a column cleanup instead of an ethanol precipitation for your cut insert.

If you are not sure about the double digestion of the pET28, you can also try a sequential digestion... and make sure that the restriction enzymes are in good shape...

Also check the ligase or its buffer, they may be degraded. Also, the salts in the ligases buffer often precipitate, so you have to make sure they are very well dissolved before using. You can try adding some fresh ATP into the ligation reaction... 

I assume that you are cloning into DH5a or similar recombinase mutant strain, right?

Another thing to take into account is the quality of the competent cells, you need quite efficient ones...

Good luck

Thank you everyone for your valuable suggestion. 

I am using restriction enzyme from NEB, so both of them work in the same buffer. Will it make a different if I do a sequentail digestion in thi case?

I did check the transformation effecient it looks good and I constantly getting empty pET so i think that should not be the problem

I also tested my ligase with DNA ladder. A shift in the ladder was observed so I am suspecting of incomplete digestion  

That is why I tried to incubate it overnight

Hi Shanice, 

If you were my student, this is how you would be doing it.

1. Clone your PCR product into an intermediate plasmid, such as pGemTEasy, using TA cloning

2. digest both plasmids with restriction enzymes in a double digest (providing they are compaible in the same buffer - sequenctial digests are only useful if they are not compatible)

3. gel purify both insert and vector

4. determine concentration of insert and vector, and use a molr ratio of insert 3:1 of 5:1 (maybe higher ratio if insert is big), with 50 ng of vector

5. ligate overnight at 16C

6. transform.

By doing the initial ligation into a random plasmid, you will find they restriction digestion efficiency to be much higher than directly cutting the PCR product. You can also sequence that product, and make sure it is correct before proceeding any further - you'd hate to get to the end only to realise it was wrong. By doing the gel extractions, you can make sure you get the right product, and it helps filter away non-specific products. By measuring the concentrations, you know EXACTLY what is going into each reaction. Ligating overnight will improve efficiency. Dont bother running the ligation mix on a gel - it wont tell you anything constructive.

I hope that helps somewhat. If you want some more specific feedback, it might be helpful to outline some of your actual protocols.

Cheers, 

Steve

when we have have problems like this we switch to Infusion cloning (which you dont need to ligate), I am attaching the manual,

Hi,

your cloning stratery seems right but this problem may occur in few specific clones due to the reasons unknown. I have come across with the same situation at least in 2-3 times and i SOLVED  the same just by changing the cloning host from DH10B to Genehogs cells. 

Overall, I used the same set of enzymes and single RE buffer system for overnight digestion at 37 C, gel purified the fragments,  ligated with double the concentration of ligase (regular T4 DNA ligase from NEB)  for overnight at 16 C, and then transformed to Genehogs cells, and got the desired clones. Hope this helps.

Good luck

Sushanta

Hi,

I did some cloning with similar strategy of yours and it always working fine. Here is some suggestion from me.

1. Check all the sequences especially in the overhang primer site and restriction site of your restriction enzymes. I know you probably did it but worth to double check.

2. If everything is correct, basically there is nothing wrong with your procedure. My guess is that your ligation efficiency quite low. So what you can do to increase it is by dephosphorylate the vector backbone to avoid the recircularization.

3. Calculate your ligation reaction properly, ratio of the vector and insert is matter here. Try 1:5 vector:insert for the start, always work in my experience.

4. Do the ligation overnight in 16C to get optimum efficiency. Make sure that your ligation kit allowed for overnight ligation.

5. Transform all the plasmid to the competent cell, use several tube of competent cells if necessary. The point is to get all the ligated product even if it is few.

Good luck

I wouldn´t do an overnight digestion for cloning. I would try a 2-hour digestion. In my experience, gel purification reduces cloning efficiency. But I would do a phenol purification of the digested fragments before doing the ligation. Ligation overnight at 16oC. Use a new ligase buffer. Freeze/thaw of this buffer leads to lower ligation efficiency.

First of all: XhoI has inhibition by methylation, check this point. If this is not the problem, I recommend you to do what Steven R Doyle has told you about cloning your insert into an intermediary vector because: (1) you won't repeat the PCR (you'll use less enzyme), (2) you can sequence your product so you can check if there has been any mutation and (3) everything is cleaner. If you are using Pfu enzyme use pCR-Blunt.

I've just finished one of this awful cloning periods more or less like yours so, my advices are:

Good luck!

Shanice, I am sure you got plenty of good answers. I would urge you though to incorporate the following controls to your experiment, that would help troubleshoot it.

1. digested Vecor only

2. Vecor + Ligase

3. Vector + Ligase + kinase

4. non- digested vector

5. Your experiment Vecor + insert + ligase (3:1 molar ratio)

Generally you won't need more than 50 ng vector, and not more than 2-3 ul for transformation, provided your competent cells are good 

After doing all these controls it still might be that your insert is producing toxic protein and kills the host, but there is no way of knowing beforehand.

Good luck

Hi Shanice,

I haven't gone through all answers, so this may be duplicated:

I miss in your description ta´hat you purify both the digested PCR fragment and vector, instead you write "ethanol precipitation". This results in several problems: first of all, you still have your polylinker fragment in the reaction which may ligate instead of the intended new fragment. EtOH ppt without e.g. phenolizing the reaction mixture might precipitate enzymes/ proteins (BSA?) from the digests, which may interfere with an efficient dissolving of your DNA afterwards (since you get protein/ nucleic acid co-ppt).

Maybe this helps togehter with other good hints I hve seen,

Tony

I fully agree with Dr.Schaffner, EtOH precipitation without phenolizing results in many problems as he has rightly pointed out. Finally, I would add that you check the ligase enzyme your are using and also the concentration. May be by changing the brand of the enzyme or by increasing the concentration etc. might give you the desired results.

Thanks Dr Tony for pointing out this problem. But what is the best way to purify the dna after digestion? Is it enough to just use a pcr purification kits

If the excised fragment or cleaved-off part is too small to bind to the qiagen column (e.g. well below 50 bp) then this might be fine. Otherwise, separate on gel and use Qiagen or even a low-melting agarose to use the fragment directly from the molen agarose.

Good luck,  Tony

Normally, I gel purify the PCR fragment, or purify using a Quiagen kit,  then do double digest, gel purify again and then do the ligation. It works very well.

Good luck,

Dilip

a big thanks to everyone's valuable suggestion. I have finally get the cloning done. 

So my modifications include:

1. Doing a sequential digestion and use a higher XhoI concentration (it doesnt cut circular plasmid well)

2. Column purify after restriction digest