Filename: LIGATION.doc
Written by: R.Johnson (11/4/97) 1
I In Gel Ligation
1.
Heat the excised DNA fragments from a low melting sea plaque agarose gel (vector and insert) at 65 o
C until the gel melts (dilute to less than 1% agarose if necessary).
2.
To a 1.5 ml tube, add vector, insert, and water such that the total volume totals 53 µl.
Mix and heat at 65 o C. The reaction should contain 3X molar ratio of insert to vector. A mock tube containing no insert should also be prepared. It is also good to include a positive control.
3.
Prepare the ligation mix with 10 µl of 10X ligation buffer, 2 µl ligase (2 weiss units), and
35 µl ice cold water for each ligation sample. Mix.
4.
Add 47 µl of ligation mix to the melted agarose (100 µl final volume). Mix, and leave at room temperature overnight.
II Transform In Gel Ligations
1.
Remove previously prepared competent cells from freezer and thaw on ice for 20 min.
Indicate cells used
2.
Meanwhile, melt ligations at 65 o
C.
3.
Make 200
 l aliquots of thawed competent cells. Prepare one tube for mock ligation, ligation, and one for positive control. While vortexing 200 µl of thawed cells, rapidly add 25 µl of the ligation mix. Incubate sample on ice for 30 min.
4.
Heat shock sample at 37 o
C for 90 sec. Add 800
 l LB and place on shaker for 1 h.
5.
Plate 100
 l ligation sample on LB plates using the appropriate antibiotic. Spin down the remainder, remove most of the liquid suspension and plate cells from remaining 900 µl onto another plate.
6.
Incubate plates at 37 o C overnight and screen for desired insert. Record the number of colonies on each plate.
Chemical
10X ligase buffer
Vendor
Boehringer Mann.
Catalog #
1243-292
10X ligase buffer
10X ligase buffer
LB capsules
Ligase
Seaplaque agarose
T4 Ligase
T4 Ligase
New England Bio.
GIBCO/BRL
Bio101
Boehringer Mann.
FMC
NEW England Bio.
GIBCO/BRL
NA
Y90001
3002-031
481-220, 713406
50112
202L
15224-017
Eipper/Mains Protocol Manual

Filename: LIGATION.doc
Written by: R.Johnson (11/4/97)
Solutions:
LB media
LB-AMP Plates
Special notes, common problems, and troubleshooting :
If ligation is unsuccessful include the following controls
1. cut vector, remove 1 µl for transformation: shows vector is cut
2. cut vector, removed 1 ul, ligate,transform: shows ligation works
Did you use the correct antibiotic on plates?
Agarose Ligation efficiency data
Seaplaque < 0.5% in final ligation
Nusieve <1.5% in final ligation
2
Eipper/Mains Protocol Manual