Definitely when increasing the temperature the rate of reaction will Increase. Before going for 4 degree Celsius overnight incubation better to perform 1 1/2 to 2 hour incubation at Room temperature (25 degree Celsius) and keep in 4 degree Celsius. Keeping more time in higher temperatures may reduce the efficiency since ATP was there in the ligation mixture and DNA was more prone to degrade at their sticky ends. 

Definitely not!! pGEM-T cloning relies in the formation of two A=T bonds that I'm not sure can be formed at 25ºC or at 16ºC. If you want to increase cloning yield you can try the following:

- ligate at 4ºC for extended periods of time, up to overnigt.

-reduce ligation volume to 5 ul (yes! it can be done)

-optimize the heat-shock (tempetarure/time). Different heat-shock-times at the same temp. and choose the one that gives you more colonies

In general it is convenient to work out this reaction the first time you do it. You know, use always the same kind of tubes, the same water bath, etc. small variations in the conditions can cause big variations in yield. And at the end, think that you only need ONE colony, so don't be too paranoic with regard to the yield.

Good cloning!!

Estanis

It has indeed worked for me at 16C O/N. Although I cannot comment on whether it has worked better than 16C 4hours or not since I found similar results. You can go ahead and try that protocol. If you have planned for ligation after A-tailing then I suggest you use minimal quantities of dATP since it inhibits ligase. Also, ensure that you have high effiency competent cells (106) when you transform the ligation mixture. Also as suggested by Estanis you can redyuce the ligation volume. That too certainly helps.

Another critical point is, as indicated by Mallika, the quality of competent cells. There are commercial competent bacteria that are up to 10e8 colonies/ug supercoiled plasmid. This makes a big difference!!!

Thanks all. I have contacted Promega and their scientist assured me that the ligation can be done overnight at 16 degree celcius. But they suggested to try 1 hour incubation at room temp and overnight at 4 degree celcius to optimize the ligation reaction. I tried the suggested procedure and I have much better success now. Thanks for the suggestions guys!