Day 3: Ligations


 This protocol is for ligating genomic DNA fragments into pGEM-T Easy Vector (from Promega). The pGEM-T vector has already been linearized by digestion with the restriction enzyme EcoRV and a 3' terminal thymidine is added to both ends. These single 3'-T overhangs at the insertion site greatly improve the efficiency of ligation of DNA inserts into the plasmids by preventing recircularization of the vector and providing a compatible overhangs for DNA inserts. We will to add a single deoxyadenosine tail to the 3' ends of the DNA inserts before we ligate the inserts into the plasmid vector to increase the efficiency of the ligation.

I.    DNA inserts tailing procedure (for the whole class):
 

1. Estimate the concentration of your DNA inserts. This can be done either using a spectrophotometer or by visual inspection of the agarose gel that your TA run. Dilute your DNA inserts to 100 ng/ul

2. Heat about 200 ng DNA inserts at 95 oC for 10 min.

3. Add 15 ul of dATP (2 mM stock solution) and 5 unit of Taq DNA polymerase.

4. Incubate at 70 oC for 15 min.

5. Keep the inserts on ice.
 

 II.    Ligation procedure (for the whole class, each group will do 1-2 tubes): 1. Briefly centrifuge the pGEM-T Easy Vector and Control Insert DNA tubes.

2. Set up the ligation reactions. We will set up a total of 5 tubes per group: 3 standard reaction tubes, one positive control tube and a background control tube. Use 0.5 ml tubes.

 Reaction #1: insert: vector molar ratio =3 :1
Reaction #2: insert: vector molar ratio =1 :1
Reaction #1: insert: vector molar ratio =1 :3

A 1:1 ratio is usually best, but ratios from 3:1 to 1:3 also work well. From this molar ratio you can calculate the ng of DNA needed for the reaction. The pGEM plasmid is about 3000 bp in length, and the average length of your fragments should be about 600 bp. Therefore, the vector is five times more massive. For all reactions, we will use 50 ng of vector. To calculate the appropriate amount of DNA inserts to include in the ligation reaction, you can use this formula:

Nanogram (ng)of inserts= (ng of vector x size of insert)/size of the vector x (insert : vector molar ratio).

For example, for reaction #1, you need: (50ng vector x 0.6 kb insert)/3.0 kb vector x (3/1)=30ng insert.
 
 
 
 
Reaction1
Reaction1
Reaction1
Positive control
Background control
T4 DNA ligase 10 X buffer
2 ul
2 ul
2 ul
2 ul
2 ul
PGEM-T Easy Vector (50 ng)
2 ul
2 ul
2 ul
2 ul
2 ul
DNA inserts
X1 (ul)
X2 (ul)
X3 (ul)
-
-
Control insert DNA
-
-
-
4 ul
-
T4 DNA ligase
2 ul
2 ul
2 ul
2 ul
2 ul
DdH2O to a final volume of
20 ul
20 ul
20 ul
20 ul
20 ul

Thus, X1 =30 ng insert. Similarly, X2=10 ng, and X3= 3 ng. Your tailed DNA inserts concentration is about 10 ng/ul.

3. Mix gently by pipetting up and down several times. Incubate the reaction overnight at 4 oC.   4. Heat reaction to 70° C for 20 min to kill the ligase enzyme.   5. Add 10 µl of 5M NaCl to the 20µl ligation reaction (bringing it to ca. 1.5M NaCl). Add 2 volumes (60 µ l) 100% EtOH (room temp), vortex and place at -70C for 1 hr.   6. Centrifuge at 4° C for 10 min. Carefully pipet off supernatant and discard.

7. Add 1 ml ice cold 70% EtOH, vortex briefly and centrifuge for another 10 min. Remove supernatant and repeat.

8. Add 10 µl water to pellet and dry in speed vac. Resuspend in 20 µl sterile water. DNA is now cleaned and ready for transformation.