This cloning should be straight forward, unless you are trying to do blunt end cloning. You will probably need to elaborate on the specific problem you are facing, vector used and competent cells (type) you are using. To begin with (this is a trick I learnt from somewhere), add your vector and insert together, place on water bath at 50C for 5 min, cool down and then add your Ligase. When adding Ligase, just drop into tube and leave it, DON'T vortex or centrifuge. T4 Ligase is a very sensitive enzyme.

Next, start with a equal molar ratio of vector and insert first and then gradually increase the insert conc not the vector.

Also, 16C overnight is a decent trick, but 1 hr is usually more than enough, you can try everything if you are that desperate. Check and recheck your vector, run a gel and see if the vector is 'clean'. You should have 3 clear bands, if you have a smear, it probably means your vector has degraded.

Finally, if all else fails, and if your lab is a well funded one, you can always try commercial cloning. All you will need is to send them the vector and insert and they will clone it for you, no questions asked. I would suggest that as a last ditch effort.

This is the best I can help you for now, unless you give me some more details.

This cloning should be straight forward, unless you are trying to do blunt end cloning. You will probably need to elaborate on the specific problem you are facing, vector used and competent cells (type) you are using. To begin with (this is a trick I learnt from somewhere), add your vector and insert together, place on water bath at 50C for 5 min, cool down and then add your Ligase. When adding Ligase, just drop into tube and leave it, DON'T vortex or centrifuge. T4 Ligase is a very sensitive enzyme.

Next, start with a equal molar ratio of vector and insert first and then gradually increase the insert conc not the vector.

Also, 16C overnight is a decent trick, but 1 hr is usually more than enough, you can try everything if you are that desperate. Check and recheck your vector, run a gel and see if the vector is 'clean'. You should have 3 clear bands, if you have a smear, it probably means your vector has degraded.

Finally, if all else fails, and if your lab is a well funded one, you can always try commercial cloning. All you will need is to send them the vector and insert and they will clone it for you, no questions asked. I would suggest that as a last ditch effort.

This is the best I can help you for now, unless you give me some more details.

1 The molar ratio, vector:insert=1:2~1:5 (make the molar of insert more than)

2 When use T4 ligase, 22℃ overnight or 16℃ 1~3h.

3 You can do this experiment from the first step such as PCR~

Good luck~~

Thank you Marie and Shirlay! That was very insightful.

Marie, indeed I am doing a blunt ended cloning and to make it further challenging, I am using only one enzyme which cuts in two places. While the enzyme cut sites are at very convenient positions, the probability to self ligate is very high. So yes, I have dephosphorylated the vector.

The second paragraph of your answer suggests that I gradually increase the insert conc. So for example, I start with, lets say 2ul of insert and 2ul of vector (i know this is not the same as molar conc.) and then increase upto 6ul of insert so that alongwith 1ul of ligation buffer and 1ul of ligase I have a 10ul reaction in total. Is this right?

As is common practice, I do a visual estimate of the bands I cut out from the gel. Eg. If the insert and vector are both equally bright, I use only part of the vector band and the complete insert band. What is your opinion on this?

Finally, the vector has a ccdb cassette which means I only use ccdb survival cels for transforming.

You have to be careful with that approach as equal intensities do not mean equal quantities. We run dilutions of a known amount of DNA of the same size of insert and vector (using a ladder, for example, works well for both). Then we calculate the ng per ul for each and use the formula: ng of insert/length of insert = ng of vector/length of vector. That will give you the ng of insert needed per ng of vector for a 1:1 ratio. For difficult ligations we usually do 1:1, 1:3 and 1:9 (vector:insert).

Generally 3:1, but blunt end cloning is very inefficient. Also gel purification can contaminate samples and inhibit ligases. Try cleaning up fragments over a column. It would help if at least one end was sticky, any way to change your cloning strategy?