Building a Reporter Gene to Determine Chromatin Protein Function
Ligation of KAH182 = BBa_S04739 and KAH201 = BBa_S04745 and cloning in DH5α-T.
Assembly strategy: The backbone KAH201(S/P)/3403 , the insert is KAH182(X/P)/1675.
Digests
(1,2) KAH182, 1675 bp, Cut with Xba1 and Pst1, (3,4) KAH201, 3403 bp, Cut with Spe1 and Pst1
Plasmid DNA
30.00 μl
Enzyme 1*
1.0 μl
Enzyme 2*
1.0 μl
10x FastDigest buffer + green loading dye
3.00 μl
dH2O
0.00 μl
30.0 μl total
Incubate at 37°C for 10 minutes.
Enzyme 1 for KAH182 is Xba1 and for KAH201 is Spe1.
Enzyme 2 for KAH182 and KAH201 is Pst1.
Make the (1%) agarose glee and add 30μl of restricted vector in one well and 10 μl of ladder.
Ligations
Ligation
1. E. coli DH5α-T KAH182(X/P)/size, 15.85 ng + KAH201(S/P)/size, 36.29ng
KAH182/KAH201 2:1 No Colonies
2. E. coli DH5α-T KAH182(X/P)/size, 15.85 ng + KAH201(S/P)/size, 36.29ng
KAH182/KAH201 3:1 One Colony
3. E. coli DH5α-T KAH182(X/P)/size, 15.85 ng + KAH201(S/P)/size, 36.29ng
KAH182/KAH201 4:1 Two Colonies
4. KAH201(S/P)/size, 36.29ng (Control Plate)
No Colonies
Calculations are for the ng of insert we need to get a 2:1, 3:1 and 4:1 ratios of insert molecules to 50 ng vector molecules
1 (2:1)
2 (3:1)
3 (4:1)
4 (- Ctrl)
Insert DNA
3.11
4.66
6.21
---
Vector DNA
1.37
1.37
1.37
1.37
2x lgn buf (Roche)
5.48
7.03
8.58
5.0
T4 ligase (NEB)
1.0
1.0
1.0
1.0
dH2O
0.00
0.00
0.00
2.63
10.96 μL
14.06 μL
17.16μl
10μL
The incubation time for the ligation process was 15min at room temperature.
Fast transformation, 15 min on ice.
Confirm The Assembly
White dashed lines border show the vector backbone, 3200 bps and the KAH181+KAH201 (1675 + 226) constructed gene.Backbone, 1901 bps, Cut with EcoRI and PstI.
Plasmid DNA
2.0 μl
EcoR1
1.0 μl
Pst1
1.0 μl
10x FastDigest buffer + green loading dye
1.5 μl
dH2O
9.5 μl
15.0 μl total
Incubate at 37°C for 10 minutes.
Make the (1%) agarose glee and add 15μl of restricted vector in one well and 10 μl of ladder.
PcTF Subcloning in E. coli
