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PcTF Subcloning in E. coli
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04/18/2013
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.
Ligation |
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1. E. coli DH5α-T KAH182(X/P)/size, 15.85 ng + KAH201(S/P)/size, 36.29ng |
KAH182/KAH201 2:1 No Colonies
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2. E. coli DH5α-T KAH182(X/P)/size, 15.85 ng + KAH201(S/P)/size, 36.29ng |
KAH182/KAH201 3:1 No Colonies 3:1 1 Colony
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3. E. coli DH5α-T KAH182(X/P)/size, 15.85 ng + KAH201(S/P)/size, 36.29ng |
KAH182/KAH201 4:1 Two Colonies
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4. KAH201(S/P)/size, 36.29ng (Control Plate) |
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- 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
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1 (2:1) |
2 (3:1) |
3 (4:1) |
4 (- Ctrl)
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Insert DNA |
3.11 |
4.66 |
6.21 |
---
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Vector DNA |
1.37 |
1.37 |
1.37 |
1.37
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2x lgn buf (Roche) |
5.48 |
7.03 |
8.58 |
5.0
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T4 ligase (NEB) |
1.0 |
1.0 |
1.0 |
1.0
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dH2O |
0.00 |
0.00 |
0.00 |
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2.63
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10.96 μL |
14.06 μL |
17.16μl |
10μL
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- The incubation time for the ligation process was 30min at room temperature.
- Fast transformation, 30 min on ice.
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