Used [http://www.insilico.uni-duesseldorf.de/Lig_Input.html ligation calculator] to determine proper amounts of insert and vector.
Vector size: 2200 bp
Vector amount: 50 ng
3:1 insert to vector ratio
Insert size:
3-mer: 406 bp -> 27.68 ng
6-mer: 712 bp -> 48.55 ng
MaSp2 Seq AB: 170 bp -> 11.59 ng
ligate in 20 uL reaction
3-mer
6-mer
MaSp2 Seq AB
T4 ligase
1 uL
1 uL
1 uL
Insert
2 uL
7 uL
0.5 uL
Vector
1 uL
1 uL
1 uL
10x T4 Ligase Buffer
2 uL
2 uL
2 uL
ddH2O
14 uL
8 uL
15.5 uL
Total
20 uL
20 uL
20 uL
25 C
25 min
65 C
10 min
12 C
hold
Small Scale Liquid Culture for BBa_525998 and BL21(DE3)
set up 6 ml cultures for BBa_525998 and BL21(DE3) with chloramphenicol for BBa_525998 and no selection for BL21(DE3).
3 cultures for BBa_525998, plan to miniprep and sequence verify.
1 culture for BL21(DE3), plan to make chemically competent cells.
Drop Dialysis
Performed drop dialysis of 2 uL of ligation reactions against ultra-pure water for 10 minutes to remove salts in preparation for electro-transformation.
Three drops on same membrane.
Electro-Transformation
Electro-transformed the 3-mer, 6-mer, and MaSp2 Seq AB from the drop dialysis. Pulse times listed below.
