Revision as of 23:02, 13 July 2012 by Gutiloluis (Talk | contribs)



Bacterial DNA extraction protocol

  1. Shaking culture overnight.
  2. Centrifuge twice at 10,700 rpm in 1.5 ml eppendorf 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 of NaCl 1M and Resuspend.
  4. Centrifuge 2 minutes at 10,700 rpm and remove the supernatant.
  5. Resuspend the pellet on 567 uL



Electro-competent cells of E. coli DH5α are electroporated at 1.25 mV, immediately we add SOC medium to the bacteria and we incubated on a shaker at 37°C for one hour, then, we culture cells in a solid medium containing the antibiotic of selection.

Primer Design

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
Total 25

PCR - Taq DNA polimerase

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
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 strain and resuspend the cells taken on 50 μL of H2O, then, put at 95°C for 10 minutes.