bhassel at orion.rz.mdc-berlin.de
Thu Jul 6 07:02:50 EST 1995
Dear Tim, 6.7.95
> 1) Am I using too much or too little DNA?
The whole mass of DNA (Vector + Insert) doesnt have to be
more than 8 ng/ul or 80 ng in a 10 ul assay.
Even with 5 ng in a 10 ul Ligation assay you can obtain
20 to 50 colonies.
A well working value is 20-50 ng total DNA mass in a
10 ul assay.
> 2) Should I clean my DNA from LMP gels? I have
> tried using DNA in LMP and cleaning it w/ a promega
> kit (bad yield)
I cut about 5 ug of DNA for the insert and 5-10 ug of
vector. Then the vector DNA is treated 10 min 65 degr.,
EtOH precip, CIPped (without prior phenol).
DNA is run on a usual TAE gel and bands
extracted with glassmilk. Its pure enough.
> 3) I am using 400U/ul T4 ligase (NE Biolabs), has
> anyone else had any problems w/ their ligase or
> 4) Will spiking my buffer w/extra ATP help & what is
> the deal w/ PEG,does it increase ligation
I think it does not.
> 5) How can I increase the competency of my
> DH5-alpha cells,Iam currently using a 50mM
> CaCl2 2X wash of cells at .3-.4OD @ 590nm
With the method used in our lab (Hanahan) we get
transformation efficiency of about 10 Mio / ug of
pBLUESCRIPT in DH5alpha. If you want the protocol,
drop a line.
Example of a ligation with different reaction times:
20 ng of pBLUE * EcoRV * CIAP (3kb)
30 ng of a 1,2 kb EcoRV Fragnment
ligation buffer (= Maniatis 2nd issue page 1.68)
1 mM ATP (end concentration)
1 u of T4 DNA Ligase (Gibco)
---------------------total volume 10 ul.
ligated at room temp,
trafo 2 ul of ligation after
10 min ---> 222 colonies (2/5 of transformation plated)
35 min ---> 377 col. (2/5 of transformation plated)
73 min ---> 624 col. (2/5 of transformation plated)
22 h ---> 680 col. (2/5 of transformation plated)
testliga without insert -> 22 col. (after 22 h)
Hope that helps
Burkhard Hassel (bhassel at orion.rz.mdc-berlin.de)
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