Haynes:PCR clip clone: Difference between revisions
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<center><font size=4>''' | <center><font size=4>'''PCR, Clip, and Clone'''</font><br> | ||
by Karmella Haynes, | by Karmella Haynes, 2015 | ||
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'''Phusion PCR''' | |||
# Amplify the insert(s) with primers that have the appropriate restriction sites on the ends. See Table 1 for recommended sequence extensions | |||
# Set up a Phusion PCR reaction as shown in Table 2. Use the tamplate table to record your work in your notebook | |||
{| {{table}} border="1" cellspacing="3" <!-- Phusion PCR table --> | |||
|- valign="top" | |||
| bgcolor=#cfcfcf | Reagent | |||
| bgcolor=#cfcfcf | Volume | |||
| rowspan="7" | <u>Expected:</u><br>1. Insert 1 = ### bp<br>2. Insert 2 = ### bp<br>3. etc. | |||
| rowspan="7" | [[Image:KAH041715_gel1.jpg|200px|Hover name]]<br>Example Image from 04/17/15; 5 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | |||
|- | |||
| DNA(plasmid) || 0.5 μL | |||
|- | |||
| 10 μM F primer || 1.0 | |||
|- | |||
| 10 μM R primer || 1.0 | |||
|- | |||
| 10 mM dNTPs || 1.0 | |||
|- | |||
| Phusion Pol. || 0.5 | |||
|- | |||
| 5X HF buffer || 10.0 | |||
|- | |||
| dH<sub>2</sub>O || 36.0 | |||
|- | |||
| || 50.0 || | |||
|} | |||
Program: Phusion (block B) | |||
* 98°C, 3 min | |||
* 35x[98°C, 10 sec; 66°C, 30 sec; 72°C, 60 sec] | |||
* 72°C, 10 min | |||
* 4°C ∞ | |||
Conclusion | |||
* All four worked. Proceed to next step | |||
Revision as of 16:53, 5 May 2015
by Karmella Haynes, 2015
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.
Phusion PCR
- Amplify the insert(s) with primers that have the appropriate restriction sites on the ends. See Table 1 for recommended sequence extensions
- Set up a Phusion PCR reaction as shown in Table 2. Use the tamplate table to record your work in your notebook
| Reagent | Volume | Expected: 1. Insert 1 = ### bp 2. Insert 2 = ### bp 3. etc. |
 Example Image from 04/17/15; 5 μL/lane; 1% agarose; Ladder |
| DNA(plasmid) | 0.5 μL | ||
| 10 μM F primer | 1.0 | ||
| 10 μM R primer | 1.0 | ||
| 10 mM dNTPs | 1.0 | ||
| Phusion Pol. | 0.5 | ||
| 5X HF buffer | 10.0 | ||
| dH2O | 36.0 | ||
| 50.0 |
Program: Phusion (block B)
- 98°C, 3 min
- 35x[98°C, 10 sec; 66°C, 30 sec; 72°C, 60 sec]
- 72°C, 10 min
- 4°C ∞
Conclusion
- All four worked. Proceed to next step
- 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)
