Building a Reporter Gene to Determine Chromatin Protein Function (Ligation into the mammalian vector)
Ligation of construct BD001(KAH182-KAH201) and mammalian vector V0200 and cloning in Bl21-DE3, The new construct is named: BD002.
KAH182 = BBa_S04739 and KAH201 = BBa_S04745 and V0200 = BBa_J176121.
Assembly strategy: The backbone V0200(N/S)/4442 , the insert is BD001 (KAH182-KAH201) (N/S)/1901.
Digests
(1 & 2) BD001 (KAH182-KAH201), 1901 bp, (3 & 4) V0200, 4442 bp, 1, 2, 3 and 4 are cut with Spe1 and Not11
Plasmid DNA
15.00 μl
NotI
1.0 μl
SpeI
1.0 μl
10x FastDigest buffer + green loading dye
3.00 μl
dH2O
10.00 μl
30.0 μl total
Incubate at 37°C for 10 minutes.
Ligations
Ligation
1. E. coli BL21 BD001 (KAH182-KAH201) (N/S)/size, 21.00 ng + V0200(N/S)/size, 43.00ng
KAH182/KAH201 3:1 Six Colonies
2. E. coli BL21 BD001 (KAH182-KAH201)(N/S)/size, 21.00 ng + V0200(N/S)/size, 43.00ng
KAH182/KAH201 4:1 One Colony
3. E. coli BL21 BD001 (KAH182-KAH201) (N/S)/size, 21.00 ng + V0200(N/S)/size, 43.00ng
KAH182/KAH201 5:1 One Colony
4. E. coli BL21 BD001 (KAH182-KAH201)(N/S)/size, 21.00 ng + V0200(N/S)/size, 43.00ng
KAH182/KAH201 10:1 One Colony
5. V0200 (N/S)/size, 43.00ng (Negative Control Plate)
No Colony
Calculations are for the ng of insert we need to get a 3:1, 4:1, 5:1 and 10:1 ratios of insert molecules to 20 ng vector molecules
Reactions
1 (3:1)
2 (4:1)
3 (5:1)
4 (10:1)
5 (- Ctrl)
Insert DNA
1.22
1.63
2.04
4.08
---
Vector DNA
1.62
1.62
1.62
1.62
1.62
2x lgn buf (Roche)
5.00
5.00
5.00
5.70
5.00
T4 ligase (NEB)
1.0
1.0
1.0
1.0
1.0
dH2O
1.16
0.75
0.34
---
2.38
10.00 μL
10.00 μL
10.00μl
12.40μL
10μL
The incubation time for the ligation process was 15min at room temperature.
Reaction number 1, 2, 3, 4, 5 40μL BL21 in 2.0 mL tubes; ice 2 min.; 42°C 45 sec.; add 800 μL SOC medium; shake @ 37°C 60 min.; pellet @ top speed 3 min.; resuspend in 100 μL amp liq. medium; plate on amp agar.
Confirm The Assembly
Two seperate bands in each column show the vector backbone and the insert, 4442 bps and the BD001 (KAH201+KAH182) (1901) constructed gene.Backbone, 1901 bps, Cut with SpeI and NotI. First column: Ladder, columns 1-5 are 3:1 ratio colonies, column6 is 4:1 ratio, column 7 is 5:1 ratio and column 8 is 10:1
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
Main project page
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