RESTRICTION ENZYME DIGESTS + LIGATIONS

Test digests:

20ml 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.

2ml DNA

2ml Buffer (e.g. Buffer 1,2,3,H,K,etc)

0.2 ml BSA

14.8 ml ddH2O

1ml 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

10ml total volume; 1ml DNA + 9ml Master Mix (of enzymes and stuff)

Master Mix (1X):

1ml buffer

0.1ml BSA

7.8ml ddH2O

0.1ml enzyme

i.e. for 30 samples (all being digested with the same enzyme), make a 35x MM

Regular Digests:

100ml total (DNA + restriction enzyme + other stuff)

If you are going to CIP treat it, add 71ml 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.

15ml DNA

10ml Buffer

1ml BSA

72ml ddH20 (or 71, if CIP treated)

2ml enzyme

Place in 37° water bath 2-3 hours. If you are CIP treating it, add 1ml CIP (calf alkaline intestinal phosphatase - under "C" in enzyme box) during the last hour of incubation. Run a little on gel (10ml, 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. 250ml DNA + 250ml 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 = 25ml, in this example

(at least 2.5x vol DNA) 100% EtOH = at least 625ml EtOH

0.5ml 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 100ml of 70% EtOH

9.  Centrifuge 5min.

10.  Decant and dry pellet completely.

11.  Resuspend in 10ml 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 (-).

1ml 10X T4 DNA Ligase Buffer (in my freezer box, if it has precipitate, warm in incubator)

5ml ddH20

2ml oligo (kinase digested) - add this only to your experimental (+) tube

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Heat in 65° water bath for 2 minutes

Add:

1ml previously digested DNA (i.e. pGEM/Sca1) that has been CIP treated (if applicable)+ ØCCl3

1ml 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.