(b) Then slowly pull down the droplet with the pipette tip.
(c) When the round droplet touches the DNA pellet, it will
spread and run to the bottom of the tube slowly.
(d) Repeat this process several times until there is no DNA left
(droplet stays round).
(e) Transfer the solution to the next tube.
13. Determine DNA concentration by NanoDrop.
14. Test ligation efficiency by Hind III digest of 100 ng of DNA in
a 10-μL reaction (see Note 7 and Fig. 3).
3.1.3 Electroporation
1. E. coli MAX Efficiency™ DH10B™ Competent Cells (Invitrogen) are thawed on ice.
2. 4 μL ligated PCR product are mixed with 40 μL Competent
Cells in a 1.5-mL reaction tube.
3. Incubate 1 min on ice.
4. Transfer 40 μL to the bottom of a 0.2 cm electroporation
cuvette (BioRad).
Table 2
Composition of the inverse PCR mix
ddH 2 O
41 μL
Buffer expand high fidelity (10Â)
5 μL
Volume of each primer (100 μM)
0.5 μL
dNTP mix (10 mM)
1 μL
Template (~400 ng μL
À1 )
1 μL
Expand polymerase (3.5 U μL
À1 )
1 μL
Total volume
50 μL
Table 3
Cycles for the inverse PCR
Time (min:sec)
Temp.
# Passes
Denat.
1:00
95
C
1
Denat.
0:20
95
C
1 0
Anneal.
0:30
65
C
1 0
À1
C/cycle
Elong.
5:00
68
C
1 0
Denat.
0:20
95
C
2 5
Anneal.
0:30
55
C
2 5
Elong.
5:00
68
C
2 5
A Directed Evolution System for Lysine Deacetylases
327