3 replies to this topic

[scogne]

Hi,
I used to do this a few years ago with this procedure:

5X ligation Buffer:- 250 mM Tris.Hcl (pH 7.6)
- 50 mM MgCl2
- 25 % PEG 8000 (from Sigma # P2139)
- 5 mM ATP
- 5 mM DTT

Use one unit of T4 DNA ligase and a ratio Vect/Insert ends=3
Ligate a few hours @ RT, or alternatively begin @ 15°C in a cold bath, and let it reach RT Overnight.

Have Fun...

[fly]

Many thanks for your help.
Would you let me know:
what does 5x buffer mean, does it contain half of what the 10x buffer has? I have only 10x buffer, how could i make 10x into 5x buffer?

[kimquinn]

For 5x buffer, dilute (for example) 100mL by adding 400mL of water to make a working solution of 500mL.

100mL of a 10x buffer will produce 1000mL of working buffer when 900mL of water is added.
Therefore to make 500mL of working buffer from a 10x stock, use 50mL of concentrate and add 450mL water. 10x stock buffer is twice as concentrated as the 5x stock buffer.

In my experience some 5x stock buffers may be preferable to 10x as they are less likely to form precipitates on storage.

[scogne]

Hi,

to make this 5X buffer dilute the 10X twice, don't forget PEG 8000 up to 25% in the latter.