4/30 Double digest of Honeybee PCR product and ligation into PSB1C3 plasmid backbone
Vinson and Olivia already prepared ecor1 and pst1 digested psb1c3, (35 ng/ul in honeybee box) which I will be using for ligation. It is column purified as well.
I am double digesting both the plain silk construct, as well as the promoter+silk construct with ecoR1 and pst1.
Digestion Rxn (50 ul)
One for silk and One for prom+silk
EcoR1 (C)
1 ul
Pst1
1 ul
NEB buffer 2.1 (10x)
5 ul
Silk DNA / PRom+Silk DNA
8.7 ul / 7.2 ul
H20
34.2 / 35.8 ul
Digestion incubation specifications: 37 degrees for 1 hour, and 80 degree heat inactivation for 15 minutes, hold 10C
Ligation Reaction into PSB1c3 vector
20 ul ligation reaction according to protocol using T4 ligase
2 reactions, (1 for silk, 1 for promoter + silk)
Both are at a 3:1 molar insert:vector ratio.
10X T4 Ligase Buffer
1 2 ul
PSB1C3 (50 ng)
1.4 ul
silk construct / prom + silk construct
1.4 ul / 1.06 ul
T4 Ligase
1 ul
H20
14.2 / 14.54
Reaction conditions: 16 C for 12 hours, 65 C for 15 min, hold at 10 C
