6 replies to this topic

[fly]

I want to clone a insert with a blunt and coadhesive end into PGEX-2T. I uesd the protocol in Promega T4 ligase insert without addition of PEG, and, however, I did not get any desirable transforminants. After I  excluded other causes, i plan to use PEG8000 to improve my ligation. Could you tell me your experience and recommend a quality product?

[scogne]

   Member
                                           

                          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...

[Svengali]

how long is your sticky end?

[Svengali]

and how GC rich is it? in fact, what is the restriction site?

[fly]

they are EcoR I and Sma I.
Thank you in advance.
I plan to use 5% PEG (Amersco).  Invitrogen recommends 5% PEG, 14 degree and 24 hour in 20 microliter.

[fly]

HI
they are EcoR I and Sma I.
Thank you in advance.
I plan to use 5% PEG (Amersco).  Invitrogen recommends 5% PEG, 14 degree and 24 hour in 20 microliter. What is your advice?

[Svengali]

the reason a low temperature is used for ligation is to ensure that the sticky ends anneal efficiently, not because the DNA ligase is unstable.  If you had a gc rich sticky end, (which you don't) you could have raised the temperature to 37 and the enzyme would work more efficiently.  As it stands, you could still try.  Depending on how the blunt end was produced, phosphorylating the blunt ends can help them find each other better.  For this you use polynucleotide kinase or PNK(I think) which is ATP dependent.
PEG artificially concentrates the sample but leaving the volume constant by occupying space in the solution.  This will have the effect (so the hypothesis goes) of increasing the chance that the ends will find each other.
Good luck, and check out Current protocols (roger Brent), it give full descriptions of this kind of stuff.