Haynes:PCR clip clone: Difference between revisions
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(New page: ---- '''Ryan - Receiver plasmids, assembly''' * Stage 1 - insert Regulator ORFS ** DONE - Phusion PCR-amplify ORFs (add 5'-E, 3'-X cut sites) ** '''Insert ORF(E/X) into Vector (E/X). see [...) |
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<center><font size=4>'''Type IIS Assembly'''</font><br> | |||
by Karmella Haynes, 2013 | |||
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Principle: The insert is PCR-amplified using an error-reducing, high-fidelity polymerase (Phusion) and primers that add restriction sites to each end of the insert. The PCR product is cleaned up using a DNA-binding column. A predetermined amount of clean DNA is cut at the ends (or "clipped") and the 5'phosphates are removed in a 20 μL-reaction. Enzymes are deactivated at 75°C and a small amount of the reaction is used directly as insert in a ligation. | |||
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Revision as of 16:46, 5 May 2015
by Karmella Haynes, 2013
Principle: The insert is PCR-amplified using an error-reducing, high-fidelity polymerase (Phusion) and primers that add restriction sites to each end of the insert. The PCR product is cleaned up using a DNA-binding column. A predetermined amount of clean DNA is cut at the ends (or "clipped") and the 5'phosphates are removed in a 20 μL-reaction. Enzymes are deactivated at 75°C and a small amount of the reaction is used directly as insert in a ligation.
- Clean up PCR products
- Zymo clean and concentrator
- Elute w/ 25 μL dH2O
- Assemblies
- AubR/MRV: AubR-PCR(E/X)/851 + MRV(E/X)/3201
- BjaR/MRV: BjaR-PCR(E/X)/776 + MRV(E/X)/3201
- BraR/MRV: BraR-PCR(E/X)/776 + MRV(E/X)/3201
- RpaR/MRV: RpaR-PCR(E/X)/779 + MRV(E/X)/3201
- Digests
- E/X
- AubR, 851 bp
- BjaR, 776 bp
- BraR, 776 bp
- RpaR, 779 bp
- Modular Receiver Vector (MRV)
Reagent | Rxn 1-4 | Rxn 5 |
DNA | 10.0 | 5.0 |
10X buffer | 3.0 | 3.0 |
EcoRI | 1.0 | 1.0 |
XbaI | 1.0 | 1.0 |
dH2O | 15.0 | 20.0 |
30.0 | 30.0 |
- Column clean-up
- Zymo clean and concentrator
- Measure conc.'s
Sample | OD260 | 260/280 | ng/μL |
1. AubR-PCR (E/X) | -0.005 | 5.875 | -5.001 |
2. BjaR-PCR (E/X) | -0.005 | 9 | -4.788 |
3. BraR-PCR (E/X) | -0.005 | 7.143 | -5.291 |
4. RpaR-PCR (E/X) | -0.003 | 29 | -3.052 |
5. MRV (E/X) | 0.007 | 1.388 | 7.11 |
- Note: unsure why concentrations so low. Proceed with ligations anyway, see what happens.
- Ligations
Rxn 1-4 | Neg | |
Insert DNA | 3.0 | --- |
Vector DNA (25 ng) | 3.5 | 3.5 |
2x lgn buf (Roche) | 7.5 | 7.5 |
T4 ligase (NEB) | 1.0 | 1.0 |
dH2O | --- | 3.0 |
15 μL | 15 μL |
- Transform 50 uL DH5α-turbo
- Plate on 100 μg/mL amp
RESULTS (4/22/15)
- AubR/MRV: 11 colonies
- BjaR/MRV: 11 colonies
- BraR/MRV: 6 colonies
- RpaR/MRV: 7 colonies
- MRV (neg): 3 colonies
- Success! Pick 2 colonies from plates 1-4 for streak plate and liquid cultures (5 mL each)