Protocols And Biosafety

To perform our construct the following procedures

were followed

1. Resuspension de biobricks
2. Genetic Transformation
3. Plasmid Extraction
4. Digestion
5. Band Purification
6. Ligation

Removing long plasmidial

1. Inoculate desired strains into 2 ml of Plasmid broth (see Appendix B) plus antibiotic.
2. Aerate at 30-37°C until the cultures are fully saturated ( 8 hours to overnight).
3. Pour approximately 1.4 ml of culture into a microcentrifuge tube.
4. Sediment the cells in a microcentrifuge for 30 seconds.
5. Remove the spent culture medium with a water aspirator equipped with a micropipette tip or with a pasteur pipette. Remove as much liquid as possible.
6. Resuspend the pellet in 0.1 ml of PEB (see p.502) with gentle vortexing. Make sure that the pellet is well-dispersed. Clumped cells yield decreased DNA.
7. Add 0.2 ml of ALM (see p.501) and mix gently by inverting the tube. The solution will clear
8. Add 0.15 ml of 3 M sodium acetate (pH 4.8; see p.502). Mix well by inverting and shaking the tube. A flocculent white precipitate will form.
9. Sediment the precipitate at 14,000 rpm for 5 minutes. The pellet contains chromosomal DNA and cell debris; the plasmid is in the supernatant.
10. Recover 400ul of supernatant into a clean tube containing 0.8 ml of cold 95% ethanol. The insoluble “pellet” tends to float; avoid taking big chunks.
11. Chill for 30 minutes to overnight at -20°C. A light-brown DNA precipitate will form. This is a good stopping point; the ethanol precipitate can be left indefinitely at -20°C.
12. Sediment the precipitate in a microcentrifuge at 14.000 rpm for 5 minutes.
13. Discard the supernatant and rinse the pellet with 1ml of cold 95% ethanol.
14. Sediment the pellet in a microcentrifuge at 14.000 rpm for 2 minutes and remove the residual ethanol with a micropipette.
15. Dry the pellet in a rotary evaporator for 5 minutes or in air for 30-60 minutes.
16. Dissolve the pellet in 0.05ml of TE (see p.502) and store at 4°C. Allow 60 minutes or more for the DNA to go completely into solution.
17. Add 0.05ml of 5 M LiCl (see p.502) and mix well.
18. Incubate on ice for 5-10 minutes.
19. Sediment the precipitate in a microcentrifuge at 14,000 rpm for 5 minutes. The pellet contains RNA and residual cell debris; the plasmid is in the supernatant.
20. Recover 100ul of supernatant into a clean tube containing 0.2ml of cold 95% ethanol.
21. Chill for 60 minutes to evernight at -20°C. A brown DNA precipitate will form. This is a
22. Sediment the precipitate in a microcentrifuge at 14,000 rpm for 5 minutes.
23. Discard the supernatant and rinse the pellet with 1ml of cold 95% ethanol
24. Sediment the pellet in a microcentrifuge at 14,000 rpm for 2 minutes and remove the residual ethanol with a micro-pipette.
25. Dry the pellet in a rotary evaporator for 5 minutes or in air for 30-60 minutes.
26. Dissolve the pellet in 0.05ml of TE. The plasmid DNA is now ready for restriction analysis; 5ul per digest is usually sufficient. The DNA is fairly stable at 4°C, but it can be frozen at - 20°C for long-term storage.

Short Plasmid Extraction

1. Pelleting 1ml of culture for 5 minutes
2. Remove excess. Re-suspend pellet in 250uL suspension (Cell Resuspension Solution)
3. Add 250uL of Lysis and invest 4 times .
4. Add 10ul Nuclease-Free Water solution. Invest 4 times . Leave for 5 minutes at room temperature
5. Add 350ul of Neutralization . Invest 4 times .
6. Centrifuge at maximum speed for 10 minutes at room temperature.
7. Reverse spin column in the collection tube .
8. Discard the clear lysate ( 700ul ) of the spin column
9. Centrifuge at maximum speed for 1 minute. Discard it below.
10. Stick on 750ul of Wash Solution (ethanol). Centrifuge at maximum speed for 1 minute. Discard supernatant.
11.Stick on 250uL of Wash Solution . Centrifuge at maximum speed for 1 minute. discard supernatant
12.Transfer to a tube
13.Add 80ul of H2OMQ . Centrifuge for 1 minute.

Genetic Transformation Protocol

Add 250 ul of chemocompetente bacteria to an Eppendorf tube Add 2 ul of plasmid ( BioBrick ) Take to the ice for 10 minutes. Take to thermoblock for 50 seconds at 42°C 2 minutes at 0°C Leave it for 10 minutes at room temperature Add 5 ml in a Falcon tube (15 ml ) and 5 ul of antibiotic Insert the material from Eppendorf tube to falcon tube. Incubate 24-48 hours at 37 ° C Genetic transformation (according to IGEM )

1. Add 50 ul of chemocompetente to an Eppendorf tube
2. Add 2 ul of plasmid (mix with little tap)
3. Stand on ice for 20-30 minutes.
4. 42°C heat shock for a minute
5. Take to the ice for 2 minutes
6. Insert 200 ul of LB
7. Take it to thermoblock at 37 ° C for 20-30 minutes.
8. Add 5 ml of LB to a Falcon tube (15 ml) and lead plate with 5 ul of antibiotic
9. Insert the the eppendorf tube material to falcon tube
10. Incubate at 37 ° C for 24-48 hrs

Double Digestion

Blend:

2 ul buffer 1
2 ul buffer 2
1 ul enzyme 1
1 ul enzyme 2
0.5 ul BSA
10 ul of plasmid
3.5 ul H2O MQ
Incubate at 37o
C for 30 minutes

Digestion

Blend:

2 ul Buffer 1
2 ul Buffer 2
1 ul enzyme
1 ul enzyme
0.5 ul BSA
10 ul plasmid
4.5 ul H2O MQ
Incubate at 37o
C for 30 minutes

P R O T O C O L

DNAPurificationbyCentrifugation

GelSlice andPCR ProductPreparation

A. Dissolving the GelSlice

1. Followingelectrophoresis,exciseDNAband from gelandplace gel slicein a 1.5ml microcentrifuge tube.
2. Add 10µl MembraneBindingSolution per 10mg of gel slice.Vortex and incubate at 50–65°C until gel slice is completely dissolved.

B. Processing PCR Amplifications

1. AddanequalvolumeofMembraneBindingSolutiontothePCRamplification. Binding of DNA
1. InsertSVMinicolumn intoCollectionTube.
2. TransferdissolvedgelmixtureorpreparedPCRproducttotheMinicolumn assembly. Incubate at room temperature for 1 minute.
3. Centrifuge at 16,000× g for1 minute.Discard flowthrough and reinsert Minicolumn intoCollection Tube. Washing
4. Add 700µl Membrane Wash Solution (ethanol added). Centrifuge at 16,000 × g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube.
5. RepeatStep4with500µlMembraneWashSolution.Centrifugeat16,000×g for 5 minutes
6. Empty the Collection Tube and recentrifuge the column assembly for 1 minute withthemicrocentrifugelidopen(or off)toallowevaporationofanyresidual ethanol. Elution
7. CarefullytransferMinicolumntoaclean1.5mlmicrocentrifugetube.
8. Add50µlofNuclease-FreeWatertotheMinicolumn.Incubateatroom temperature for1 minute.Centrifuge at 16,000 × g for1 minute.
9. Discard Minicolumn and store DNA at 4°C or –20°C

Team:Colegio EmelinaU/Experiments