RESTRICTION ENZYME DIGESTS + LIGATIONS
Test digests:
20l total (DNA + restriction enzyme + other stuff). Enzymes (e.g. HindIII, EcoR1)are
in freezer between Mark and Jen. Add your enzyme last!!!!
Figure out which buffers your particular enzyme needs by looking at the chart on Jen's
bench - these are found in the freezer between Mark and Jen.
2l DNA
2l Buffer (e.g. Buffer 1,2,3,H,K,etc)
0.2 l BSA
14.8 l ddH2O
1l enzyme (added LAST)
Place in 37 water bath (or incubator) for 2-3 hours, then run on gel to be sure digest
worked. If digest worked, DNA should ALL be linear (i.e. one band only)
Test Digest for Minipreps
10l total volume; 1l DNA + 9l Master Mix (of enzymes and stuff)
Master Mix (1X):
1l buffer
0.1l BSA
7.8l ddH2O
0.1l enzyme
i.e. for 30 samples (all being digested with the same enzyme), make a 35x MM
Regular Digests:
100l total (DNA + restriction enzyme + other stuff)
If you are going to CIP treat it, add 71l of ddH2O instead of 72 .You can't do this if your
digested DNA is not in Buffer 2,3,or 4 - the CIP doesn't work in Buffer D. CIP removes
the P, so that the DNA doesn't reanneal to itself, therefore making any future ligations
easier.
15l DNA
10l Buffer
1l BSA
72l ddH20 (or 71, if CIP treated)
2l enzyme
Place in 37 water bath 2-3 hours. If you are CIP treating it, add 1l CIP (calf
alkaline intestinal phosphatase - under "C" in enzyme box) during the last hour of
incubation. Run a little on gel (10l, to be sure that your digest worked). THEN…..
Phenol extract, and ethanol precipitate:
Phenol/EtOH ppt:
Digested DNA (CIP treated, if applicable) + equal parts Ø CCL3 (phenol chloroform, take
from bottom yellow layer) i.e. 250l DNA + 250l ØCCl3
1. Vortex to milky white/yellow
2. Centrifuge at 14K for 2 min.
3. Transfer aqueous phase to new tube.
4. To this aqueous phase of your digest, add:
(1/10 vol ) 3MnaOAc = 25l, in this example
(at least 2.5x vol DNA) 100% EtOH = at least 625l EtOH
0.5l tRNA
5. Put tube in -80freezer for at least 20min. (longer is better)
6. Centrifuge at 14K for 15min.
7. Decant EtOH
8. Add 100l of 70% EtOH
9. Centrifuge 5min.
10. Decant and dry pellet completely.
11. Resuspend in 10l TE.
12. NOW you can do ligations!
Ligations
For ligations, do an experimental tube and a control, i.e. one tube that has your oligo (+),
one without (-).
1l 10X T4 DNA Ligase Buffer (in my freezer box, if it has precipitate, warm in incubator)
5l ddH20
2l oligo (kinase digested) - add this only to your experimental (+) tube
--------------------------------------------Heat in 65 water bath for 2 minutes
Add:
1l previously digested DNA (i.e. pGEM/Sca1) that has been CIP treated (if applicable)+ ØCCl3
1l T4 DNA Ligase (in enzyme box)
Store in 15 water bath overnight.
Then transform this into bacteria, plate on LB-amp, and do minipreps to find out which
bacterial colonies contain the inserted oligo.