- You might have a problem with antibiotic selection. Do you include a negative control (plating untransformed cells onto your selection medium) in your experiments? Is this control OK (showing no colonies)?

- It could also be a contamination problem (i. e., the cells you observe are contaminants). When you prep them, do you see the empty vector at the expected size?

- Use different batches of enzymes. Sometimes they are too old or have been stored at RT for longer time by accident.

- After the ligation reaction you can check if the ligase was able to ligate your DNA fragments. Here you can find information on how this is done:

http://www.thermoscientificbio.com/whats-new-t4-dna-ligase/

If this experiment shows you that you have a positive ligation reaction then you can rule out the possibility that your ligase failed. It might at least help you coming closer to solving your problem...

Good luck!

Another possibility is that you did not allow a long enough region at the ends of your PCR product to get cutting of the restriction enzyme. Some enzymes to cut at all require 3-5 bases added at the end of the fragment prior to the restriction site. For example, if you are engineering an EcoR I site at one end, your primer may be

XXXXGAATTCXXXXXXXXXXXXXXXXXXXXXXXX.......

If your primer is

XGAATTCXXXXXXXXXXXXX..........

It may not cut at all.....again is dependent on the enzyme. The technical section of NEB website has more info.

I agree with Christopher and one way to bypass this situation is performing first a pGEM cloning and afterwards you can digest your construction with your enzymes with no problems. You have to obtain two different plasmids for one construction but it is more straightforward  

1. When you received this plasmid, did you sequence it or otherwise verify that it's what you think it is? That's my first recommendation, especially since you are digesting for 16 hours. That seems way longer than normal. I usually digest plasmids for 2-3 hours.

2. Assuming it's the right plasmid, check that each enzyme is cutting. Set up a double digest (SacI and HindIII), two independent single digests (SacI or HindIII, plus water instead of a 2nd enzyme), and a mock digest (water instead of enzyme). When I do this, I usually make the double digest a larger volume (e.g. 60 uL) and scale down the 3 controls (e.g. 15 uL reactions) to avoid wasting uncut plasmid. I incubate all 4 reactions the same way, then run ~5-10 uL (enough for 500 ng DNA) on a 0.5% agarose gel. Uncut vector runs faster/smaller because it's supercoiled. Linear vector runs true to size and looks shifted up. If all goes well, you'll see 1 lane with lower uncut plasmid band and 3 lanes with higher bands for linearized plasmid.  The mock digest lane might actually show you 3 bands (lowest and brightest is uncut plasmid, then 2 higher but fainter bands for nicked and cut plasmid). If any of your single digests are showing a mixed population, then your digestion is inefficient. Try fresh enzyme, more enzyme, or a different buffer. If any of your single digests are only showing a low band matching the lowest band in the mock digest, then you're not getting cutting. Either your plasmid sequence is wrong, your enzyme is completely inactive, or something is inhibiting the enzyme.

3. If your restriction enzymes are working, great. Now you should dephosphorylate the plasmid before your ligation step. Some people don't do this if the sticky ends are incompatible (less likely to re-circularize), but you're seeing a lot of uncut vector so I suggest you do this. Most people use calf intestinal alkaline phosphatase (CIAP) or shrimp alkaline phosphatase or antarctic phosphatase. Incubate for 30-60 minutes typically. I just add the phosphatase enzyme directly into the restriction digest reaction near the end without any intermediate heat inactivation or clean up, e.g. 2.5 hour with restriction enzymes, add CIAP, incubate additional 30 min with all 3 enzymes. Now gel purify DNA to remove proteins.

[Warning: never dephosphorylate the insert! The insert needs a 5' phosphate for the ligation to work. You don't need special modified primers. If you're cutting a PCR product with restriction enzymes, you're generating the 5' phosphate you need. Your insert should be fine based on what you've written.]

Thx Christian Q. Scheckhuber for your explanation. But my control show no colonies so i'm sure my antibiotic ok.

Mr  Christopher A Seid  and Mrs. Ana Camacho i don't think my primer was wrong. but thx for your explenation

Mrs Tania L Gonzalez , i digest my plasmid until 16 hours cause when i digest 2-3 hours, not all of them was digest. there is some uncut plasmid.

this my photo ligation result. My interest gene 1,4kb and pEX18Gm 5,8kb.

But as you can see, there are many band are showed. I guess my gene interest was ligated.

H5: ligation vector:insert =1:5

H10:ligation vector:insert =1:10

HP: self lgation vector without insert

Hi Heny,

Please check your restriction site for SacI and HindIII  the end of your PCR product (stickyyends) whether they are in the same manner 5'-3 in the vector cloning site where you want to clone. Just check them first and continue that's my suggestion.

Dear Mr. Heny, 

My opinion is

1- check the restriction enzymes buffer composition for both scal1 and Hind111 if the composition is the same or not and from the same company or not . 

2- You can use two step digestion first scaI then ethanol precipitation of the first mix  then use HindIII 

(Last month i have done the same procedure and it was successful)  

From the gel image, it appears that only inserts were ligated up (if you have insert only ligation reaction running side by side with the three lanes showing here, it will tell that your ligation reactions only happen among the insert molecules, some how the vector molecules are missing from the scene. as the gel did not show a band of insert+vector in size.

This is probably due to the enzymatic digest step, in fact, if you do double digest of these two enzymes, you will never be able to achieve complete double digest in one reaction for both HindIII and SacI (refer to NEB technical guide).

You will need to do single digest one after another after making sure that you have at least 3nt at the end before cutting site at least for HindIII.

Not too sure how you manage to have no vector in the ligation reaction.

As Ling Li has pointed out above, seems like your gel has a number of insert-insert ligation products ( bands at multiples of 1.4(kb)). Your vector/insert ratio is too low. vector/insert ratio should be not more than 1:3 for ligation reactions inorder to avoid non-specific ligated products, as in your case. Change the ratio, check for buffer compatibility for enzymes and do the digestion in the following manner( if you did not do it this way):

Digest pure vector with 1st enzyme, run on a gel (1/2 microL)and check for complete digestion.

Elute the entire single digested product following your optimised protocol. Make sure you are using a half TE buffer or nuclease free water for resuspension that has less EDTA which may otherwise hamper your RD enzyme activity.

After resuspension, perform second digestion in a similar manner.

Check on gel and go for elution. I think it should work fine.

1. Check your restriction enzyme buffer compositions. Are they compatible? If not, you will have to do a double digest in two stages with a precipitation step in between.

2. While double digesting, change the order of enzymes i.e. in one set up, use SacI before HindIII and in another set up, reverse the order. This is because some enzymes cut linearised DNA better than circular DNA

3. Change your vector:insert ratio. standardize from 1:3 to 1:5

4. Also check on gel after each step of digestion and ligation.