Biotech Glossary | Bioinformatics | Lab Protocol | Notes | Malaysia University |
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Plasmid Rescue from P[lacZ] Lines in Drosophila |
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| Overview |
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| All of the commonly used P[lacZ] vectors contain sequences that allow plasmid recovery (rescue) including genomic sequences from transformed flies. Sequences required for plasmid rescue a bacterial origin of replication and an antibiotic resistance gene. Additionally, unique restriction sites, which cut in the transformation vector only on one side or the ori/resistance sequences, are required. The second site is provided by flanking genomic DNA, so that the rescued plasmid contains DNA originating from both the transformation vector and the fly's genome. | |||||||||||||||||
| Procedure |
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1. Extract DNA from forty to fifty flies (see Protocol ID#37). 2. Check the quality of extracted DNA with desired restriction enzymes and Southern blotting (see Hint #1). 3. Digest two to ten fly equivalents of DNA with an enzyme that fulfills desired criteria for recovery (see Hint #2). 4. Resuspend the digested DNA in 20 to 50 μl of TE Buffer. 5. Use half of the resuspended DNA for the ligation (usually 2 Units ligase such as T4 DNA ligase, see Hint #3) 6. Incubate the reaction mixture for 4 hrs to overnight. 7. Remove 200 μl of the ligation mixture and add 20 μl of 3 M Sodium Acetate. 8. Mix well by inversion. 9. Add two volumes of 100% Ethanol and mix well by inversion. 10. Centrifuge in a microcentrifuge for 15 min at maximum speed to pellet the DNA. Discard the supernatant. 11. Add an equal volume of 70% Ethanol to the pelleted DNA (equal to the volume discarded) and mix well by inversion. 12. Centrifuge in a microcentrifuge for 15 min at maximum speed to pellet the DNA. Discard the supernatant. 13. Resuspend the DNA in 10 to 20 μl of TE Buffer. 14. To transform into bacterial DNA, follow the protocol for Hanahan Method of Transformation (see Protocol ID#9020). |
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| Solutions |
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| BioReagents and Chemicals |
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Tris Ethanol Chloroform EDTA Phenol Sodium Acetate DNA Ligase, T4 |
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| Protocol Hints |
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1. Two to four fly equivalents are usually enough to see a good smear on Agarose gel. 2. If several different restriction enzymes are suitable, try more than one. If possible, employ an enzyme that can be heat-inactivated; otherwise, include a phenol:chloroform extraction before DNA precipitation. 3. To avoid intramolecular ligation events, perform ligation reaction in a larger volume (200 μl is usually fine). It is not necessary to add more than 2 μl of T4 DNA ligase. |
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