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 PcTF Subcloning in E. coli
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11/16/12
- ---Karmella 13:51, 16 November 2012 (EST): Here is my suggested procedure for trying the PcTF fusion cloning...
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
| Reagent |
Volume |
|
|
| DNA (plasmid) |
25.0 μL
|
| 10x buffer |
3.0
|
| EcoRI |
1.0
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| PstI |
1.0
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| dH2O |
0
|
| |
30 μL --> 37°C/ ~30 min.
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Measure concentration(s)
| Sample |
OD260 |
260/280 |
ng/μL
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| 1. pT7CFE1-CHis (E/P) |
--- |
--- |
---
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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)
| |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8
|
| Insert DNA |
--- |
--- |
--- |
--- |
--- |
--- |
--- |
---
|
| Vector DNA |
--- |
--- |
--- |
--- |
--- |
--- |
--- |
---
|
| 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
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| dH2O |
--- |
--- |
--- |
--- |
--- |
--- |
--- |
---
|
| |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL
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- Add total ligation reaction to 30 μL DH5α-Turbo cells
- Follow routine "fast" transformation procedure
- Plate onto 100 ug/mL Amp agar plates
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PcTF Subcloning in E. coli
