PCR for gene isolation.
Obtention of Biobricks by PCR
Every part was obtained from plasmid Myc pCDNA (-) 3.1 His A.
Primers Design
Table 1 annotations.
A) Extra bases to allow cuts (salmon). B) Extra base to give space (light blue). C) Extra base to avoid methylation sites (royal blue). D) Extra base to avoid methylation site or start codon (green). E) EcoRI Site (light blue background). F) NotI Site (green background). G) XbaI Site (red background). H) Pst1 Site (yellow background). I) SpeI Site (purple background). J) When it comes to coding sequences like NeoR, extra bases need to be added to ensure there is a correct space between ribosome and ATG (yellow font color with black background). K) TTA TTA (salmon font color with black background) Works as a double stop codon.
imagen
PCR’s
PCR non specific protocol
Table 1.This protocol is the New England Biolabs Taq2X Master Mix protocol.
tabla
The reaction must be gently mixed, if necessary a quick spin can collect all liquid ti the bottom.
Routine conditions for an average PCR are reported as follows:
imagen
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Gene isolation testing via digestion
All of the parts were digested with XhoI.
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Incubate at 37°C for 1:15 hours.
Inactivate at 80°C for 20 minutes.
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Experiment Three>
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Recombinant Protein Expression Protocol
Materials
Reagents
• 20 mM KH2PO4 and 10 mM KCl (pH 8) solution
• 300 mM KCl (pH 8), 20 mM Imidazole (pH 8) and 20 mM KH2PO4 solution
• Biomass recovered from the 40 mL medium
• Fresh Laemmli Buffer
• Lysozyme (20 mg/mL)
Equipment
• Centrifuge
• Micropipettes
• Conical tubes
• Centrifugal tubes
Procedure
1. Centrifuge 40 mL medium 5000 rpm, 30 min. Remove supernatant.
2. Resuspend pellet in 4 mL of a cold 300 mM KCl (pH 8), 20 mM Imidazole (pH 8) and 20 mM KH2PO4 solution.
3. Add 70uL of Lysozyme.
4. Incubate in shaker 200 rpm the biomass-lysozyme solution at 37° C for 60 minutes.
5. Centrifuge cell lysate at 14,000 rpm for 10 minutes. If the supernatant is not crystalline, the sample has to be centrifuged again. Store soluble phase at 4°C in a conical tube, covered with aluminum for chromatography.
6. Take 20 µL of the supernatant and mix with 20 µL of Laemmli buffer for SDS PAGE analysis.
7. The insoluble phase obtained from the previous centrifugation has to be washed twice with a 1% SDS solution and 20 mM KH2PO4 and 10 mM KCl (pH 8) solution. Then, mix insoluble phase with 20ul Laemmli buffer for SDS PAGE analysis.
8. Run a SDS PAGE with iGEM ITESM CEM’s protocol.
*Do not cease to contemplate that all samples should be perfectly mixed with the indicated reagents (Laemmli Buffer) before they are boiled for 5 minutes. In the case of the inclusion bodies, the boiling should be carried out for 10 minutes.
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Experiment Five
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