IGEM:UNAM-Genomics Mexico/2009/Notebook/H2 Pae/2011/07/29

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Abstract

1. I continued with the experiment of plasmid's stability of pBBR1MCS-5.

2. I continued with the conjugation of the standard pBBR1MCS-5 into R. etli.

3. I ran a Paulina's gel.

Methods

Plasmid stability

  • At 9:20 AM I took 1 ml of the culture which was, at this point, 48 hours old. I repeat the yesterday's procedure of:
1. Vortex the 1 ml culture.
2. Take 100μl of the previous culture and put them in 900 μL of liquid medium of antibiotic. This is the first dilution.
3. Vortex 30 times the dilution.
4. Repeat the steps 2 and 3 until reach the desired dilution.

I did it until reached the 8th dilution, becuase the original culture was 48 hours old and there were a lot of bacteria. Then I plaqued 100 μL of the dilutions 5, 6, 7 and 8.

  • After I took 100 colonies of the plaqued dilutions 4 and 5, which came from the 24 hours old culture; I plaqued them twice in only two petri dishes, one containing gentamicin and the other one without antibiotic. The previous in order to see how many colonies conserved/lost the plasmid.
  • At 9:20 PM I took 1 ml of the culture which was, at this point, 60 hours old. I repeat the yesterday's procedure of:
1. Vortex the 1 ml culture.
2. Take 100μl of the previous culture and put them in 900 μL of liquid medium of antibiotic. This is the first dilution.
3. Vortex 30 times the dilution.
4. Repeat the steps 2 and 3 until reach the desired dilution.

I did it until reached the 9th dilution, becuase the original culture was 48 hours old and there were a lot of bacteria. Then I plaqued 100 μL of the dilutions 6, 7, 8 and 9.

  • After I took 68 colonies of the plaqued dilutions 5 and 6, which came from the 36 hours old culture; I plaqued them twice in only two petri dishes, one containing gentamicin and the other one without antibiotic.

Conjugation

Pauilna's gel

The following is a gel (120V x 35 min) which Paulina asked me to do it.

Lanes: 

1. Isolation 15
2. Isolation 2
3. Isolation 3
4. Isolation 4
5. Isolation 5
6. Isolation 5
7. Isolation 7
8. Isolation 1
9. Isolation 8
10. Isolation 9
11. Isolation 10
12. Isolation 11
13. Isolation 12
14. Isolation 13
15. Isolation 14



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