Team:Colombia/Notebook/Protocols

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Contents

Protocols

Bacterial (Xam) DNA extraction protocol

  1. Shaking culture overnight.
  2. Centrifuge twice at 10,700 rpm in 1.5 mL tube 2 or 3 minutes. On the same tube centrifuge all the cultures and remove the supernatant.
  3. Wash the pellet twice with 1 mL NaCl 1 M and Resuspend.
  4. Centrifuge 2 minutes at 10,700 rpm and remove the supernatant.
  5. Resuspend the pellet on 567 μL TE (10 mM Tris-HCl pH 8; 1 mM EDTA pH 8).
  6. Add 3 μL proteinase K (20 mg/mL) and 30 μL SDS (10%). Resuspend gently.
  7. Incubate 2 hours 30 minutes at 37°C.
  8. Add 100 μL NaCl 5 M and 80 μL of the mix NaCl-CTAB (NaCl 0.7 M and CTAB 10%). Preheat the mix to solve it.
  9. Incubate 10 minutes at 65°C.
  10. Add 600 μL phenol.
  11. Gently shake manually for 10 minutes.
  12. Centrifuge 10 minutes at 10,700 rpm.
  13. Transfer the upper aqueous phase to a new tube and add an equal volume of chloroform-alcohol isoamyl (24:1).Gently mix by vortex.
  14. Centrifuge 5 minutes at 10,700 rpm.
  15. Transfer the upper aqueous phase to a new tube.
  16. Add 0.6 V of isopropyl alcohol and incubate at least 15 minutes at -80°C or overnight at -20°C.
  17. Centrifuge 20 minutes at 10,700 rpm. Remove supernatant.
  18. Wash the pellet with 1 mL ethanol 70% (2 or 3 times).
  19. Centrifuge 15 minutes at 10,700 rpm. Remove supernatant with pipette.
  20. Dry the pellet under vacuum for 20 minutes.
  21. Resuspend the pellet on 30 μL TE or H2O.
  22. Add 2 μL RNAse and incubate 2 hours 30 minutes at 37°C.
  23. Store the tubes at -20°C.

Miniprep

We are using the GenElute™ Plasmid Miniprep Kit of Sigma-Aldrich according to the manufacturer directions.200px|thumb|center

Preparation of electrocompetent E. coli cells

  1. Grow a colony of culture overnight on 5mL LB at 37°C shaker.
  2. Put the content of the tube on an erlenmeyer with 250mL LB.
  3. Shake at 37°C - 200rpm and continuously read the OD.
  4. When the OD reach 0,7 the cells must be kept on ice and all the implements must be cold.
  5. Centrifuge 10 minutes at 8500 rpm and 4°C. Discard supernatant.
  6. Wash the cells with deionized distilled H2O and mix by vortex. Completing then the volume of the tube with more H2O.
  7. Centrifuge 10 minutes at 8500 rpm and 4°C. Discard supernatant.
  8. Repeat steps 6 and 7 once again.
  9. Repeat steps 6 and 7 but using sterile 10% glycerol.
  10. Resuspend the product of the last centrifugation on 1mL 10% glycerol.
  11. Aliquote 40μL on Eppendorf tubes and store cells at -80°C.

Electroporation

We electroporate E. coli DH5α Electro-competent cells at 1.25 mV, then we resuspend the cells in SOC medium and incubate on a shaker at 37°C for one hour, then, we plate the cells in a solid medium containing antibiotic selection.

Primer Design

Primers were designed manually using OligoAnalyzer of IDT, secondary structures were also calculated using mfold and annealing conditions usign FAST PCR.

PCR - Pfu DNA polimerase

One reaction.

Reactives Volume(μL)
H2O 17.95
Buffer 2
MgSO4 1.6
dNTP's 0.4
Primer Fw 0.4
Primer Rv 0.4
Taq 0.06
Pfu 0.19
DNA 2
Total 25


PCR - Taq DNA polimerase Invitrogen

One reaction.

Reactives Volume(μL)
H2O 6.15
Buffer 1
MgCl2 0.5
dNTP's 0.25
Primer Fw 0.5
Primer Rv 0.5
Taq 0.1
DNA 1
Total 10

If needed add 1 μL of DMSO per reaction, and adjust the amount of H2O to a final volume of 10 μL.

PCR - Colony

Based on the protocol used with Taq polimerase, add 1 μL more of H2O and take a few cells from the strain pricking it with a toothpick and mixing them with the other PCR reactives instead of using purified DNA.


PCR - Boiling

We make a lysate of the cells and add 1 μL of that lysate instead of purified DNA using the Taq polimerase protocol. To obtain the lysate, scrape the growth solid culture and resuspend the cells on 50 μL of H2O, then, put at 95°C for 10 minutes.


Digestion of DNA with 2 different enzymes

One reaction

Reactives Volume(μL)
H2O 28,6
Buffer 4
BSA 0.4
Enzyme 1 1
Enzyme 2 1
DNA 5
Total 40

To prepare this reaction you first pipet together DNA, buffer and water, vortex the mixture and add the enzyme. Mix by pipetting and incubate for 3 hours at the temperature optimal for the enzyme (generally at 37°C).


DNA ligation with T4 DNA ligase

Reactives Volume(μL)
nuclease-free H2O 12,99
Buffer 10X 2
Vector DNA 2,5
Insert DNA 2,5
T4 ligase 0,01
Total 20

Incubate at room temperature for 16 hours.