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February 8, 2013
Type IIS Assembly - Making PCR Products
- Attempt new assembly method because piecewise assembly was unsuccessful
- Final assembly product: pSB1A3 left end / H2B / LOV +His tag/ pSB1A3 right end
- Primer design
H2B
1A3/H2B fwd 5'-cacaccaCGTCTCa TAGA ATGCCAGAGCCAGCG
H2B/LOV rev 5'-cacaccaCGTCTCa CCAA CTTAGCGCTGGTGTA
LOV
LOV fwd 5'-cacaccaCGTCTCa TTGGCTACTACACTT
LOV+his/1A3 rev 5'-cacaccaCGTCTCa TAGT ttagtggtgatggtgatgatgAAGTTCTTTTGCCGC
- First, resuspend dry primer oligos (in blue-capped tubes) to 100 μM
- Then make working solutions of 10 μM
- Reactions:
- H2B-GFP plasmid + 1A3/H2B fwd + H2B/LOV rev
- LOV plasmid + LOV fwd + LOV+his/1A3 rev
Reagents |
H2B |
LOV
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Plasmid DNA |
0.2 μL |
0.2
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primer 1 (10 μM) |
1.0 |
1.0
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primer 2 (10 μM) |
1.0 |
1.0
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2x GoTaq mix |
25.0 |
25.0
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dH2O |
22.8 |
22.8
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Total |
50.0 |
50.0
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PCR program:
- 95°C, 3 min.
- [95°C, 30 sec; 57°C, 30 sec.; 72°C, 1 min.] x35 cycles
- 72°C, 3 min.
- 4°C ∞
Expected: 1. H2B = 410 2. LOV = 461
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Results of the digests are shown.
Results of the PCR product digests are shown, with H2B on the left and LOV on the right. The gel was loaded with 5 μL samples.
- The PCR products were purified with the Zymo DNA Clean & Concentrator Kit and eluted with 20 μL dH2O
PCR Product |
260 |
280 |
260/280 |
ng/μL
|
H2B |
fill |
fill |
fill |
fill
|
LOV |
fill |
fill |
fill |
fill
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