Filename:CIPSAP.doc Written by:R.Johnson (10/18/97)
1
Protocol: Removing 5' Phosphate From Plasmid Vectors With CIP
or SAP.
I.
1.
2.
3.
4.
5.
6.
7.
8.
Calf Intestinal Alkaline Phosphatase (CIP)
Digest plasmid vector (1-10 ug) with desired restriction enzyme(s).
Check a small aliquot (200-300 ng) on agarose gel to ensure complete digestion. Add
more enzyme if digest is incomplete (Alternatively, gel purify after CIP treatment).
Clean up DNA by phenol-chloroform extractions, Gene-Clean, or similar.
If Phenol-chloroform extracts were done in Step #3 then ethanol-precipitate DNA using
1:10 v/v with 3M NaOAc and 2 volumes 100 % ethanol.
After spinning down DNA, wash DNA pellet in 70% ethanol.
Dry DNA pellet using speed vac and resuspend pellet in 20 µl of sterile water.
Set up CIP treatment in 30 to 50 µl volume using 10X CIP buffer (B-M) and enzyme as
follows:
For 5’ overhang, use 1 unit/100 pmole ends 30 minutes at 37 oC.
For 3’ overhang or Blunt, use 1 unit/ 2 pmole ends 15 minutes at 37 oC, then add 2nd
aliquot and incubate at 55 oC for 45 minutes.
Inactivate enzyme by either:
a) running immediately on agarose gel
b) proteinase K to 100 µg/ml 56 oC for 30 min in 0.5% SDS, 5mM EDTA
c) 65 oC for 60 min in 5 mM EDTA (or 75 oC 10 minutes),
For b) and c), phenol-chloroform extract and ethanol precipitate
(Note: at acid pH, EDTA will precipitate from solution at concentrations >5-10 mM).
Note:1 µg of 3 Kb plasmid = 0.5 pmole = 1 pmole ends
Alternative Quick Methods: Since Stratagene 2X has 80% activity, this can be added directly to
restriction digest. To 20 µl digest, add 2.5 µl of 100mM Tris pH 8.3, 10 mM ZnCl2. Then CIP
treat.
Eipper/Mains Protocol Manual
2
Filename:CIPSAP.doc Written by:R.Johnson (10/18/97)
II
Shrimp Alkaline Phosphatase (SAP)
Follow the initial steps 1 to 6 as described above in CIP protocol.
1.
2.
Set up digest in 20 mM Tris pH 8.0, 10 mM MgCl2, and 20 µg/ml DNA.
The amount of enzyme depends on the kind of termini and amount of DNA available.
For a typical reaction using 1 pmole of DNA termini, the following amounts are effective:
For 5’ overhang
0.1 unit
For Blunt ends
0.2 unit
incubate at 37oC for 60 min.
For 3’ overhang
0.5 unit
Enzyme dilution buffer comes with the enzyme supplied.
(Note: These are minimum effective amounts. It may be prudent to use more enzyme or longer
incubation times to ensure complete dephosphorylation. But also beware that over digestion
may also reduce the amount of good clonable vector DNA (i.e. Some percentage of vector
becomes un-ligatable).
3.
Heat inactivate SAP by 15 min incubation at 65oC. DNA is now ready to go directly into
ligations.
1 µg 1 Kb plasmid = 1.5 pmole = 3 pmole ends
1 µg 3 Kb plasmid = 0.5 pmole = 1 pmole ends
1 µg 5 Kb plasmid = 0.3 pmole = 0.6 pmole ends
Chemical
CIP
SAP
Vendor
USB
Sigma
Solutions:
Special notes, common problems, and troubleshooting:
CIP is stored at 4 oC and SAP is stored -20 oC.
References:
Eipper/Mains Protocol Manual
Catalog #
70092
M8266