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PcTF Subcloning in E. coli
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11/20/12
Assemblies : Shown as Part/cuts/purified fragment size
KAH109/pT7CFE1-CHis: KAH109/(E/P)/1132 + pT7CFE1-CHis/(E/P)/3600
KAH111/pT7CFE1-CHis: KAH184/(E/S)/1123 + "
KAH112/pT7CFE1-CHis: KAH187/(E/S)/1123 + "
KAH115/pT7CFE1-CHis: KAH188/(E/S)/931 + "
KAH148/pT7CFE1-CHis: KAH189/(E/S)/967 + "
KAH150/pT7CFE1-CHis: KAH190/(E/S)/967 + "
KAH151/pT7CFE1-CHis: KAH191/(E/S)/967 + "
Note: KAH109 is being re-done as an internal control to trouble-shoot the ligation
Note: KAH111-KAH151 are all cut and purified already
Digest (Fermentas FD)
pT7CFE1-CHis, E/P
KAH109, E/P
KAH111, E/P
KAH112, E/P
KAH115, E/P
KAH148, E/P
KAH150, E/P
KAH151, E/P
Reagent
Volume
1. KAH109, 2. KAH111, 3. KAH112, 4. KAH115, 5. KAH148, 6. KAH150, 7. KAH151 8. Human Vector PT7CFE1-CHis(3600 bp) Cut with EcoRI and PstI
DNA (plasmid)
25.0 μL
10x buffer
3.0
EcoRI
1.0
PstI
1.0
dH2 O
0
30 μL --> 37°C/ ~30 min.
Measure concentration(s)
Sample
OD260
260/280
ng/μL
1. pT7CFE1-CHis (E/P)
---
1.829
57.344
2. KAH109, (E/P)
---
2.354
36.072
3. KAH111, (E/P)
---
2.238
15.003
4. KAH112, (E/P)
---
1.862
22.857
5. KAH115, (E/P)
---
2.096
18.312
6. KAH148, (E/P)
---
2.389
23.735
7. KAH150, (E/P)
---
1.99
19.822
8. KAH151, (E/P)
---
1.90
19.701
Ligations (trouble shooting suggestions)
Note: Use 50 ng of vector this time (instead of 20)
Note: Use a 3:1 ratio of insert to vector... x μL insert = 3 * (bp of insert/ bp of vector) * 50 ng vector / (concentration of insert ng/μL)
BD-116 (KAH109)
BD-117 (KAH111)
BD-118 (KAH112)
BD-119 (KAH115)
BD-120 (KAH148)
BD-121 (KAH150)
BD-122 (KAH-151)
CTRL
1
2
3
4
5
6
7
8
Inser Name
KAH109
KAH111
KAH112
KAH115
KAH148
KAH150
KAH151
N/A
Insert DNA
1.3
3.12
2.05
2.12
1.7
2.03
2.05
0
Vector DNA
0.87
0.87
0.87
0.87
0.87
0.87
0.87
0.87
2x lgn buf (Roche)
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
T4 ligase (NEB)
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
dH2 O
1.83
0.01
1.08
1.01
1.43
1.1
1.08
3.13
10 μL
10 μL
10 μL
10 μL
10 μL
10 μL
10 μL
10 μL
Add total ligation reaction to 30 μL DH5α-Turbo cells
Follow routine "fast" transformation procedure
Plate onto 100 ug/mL Amp agar plates