User:Jorge Arturo Zepeda/Notebook/iGem LCG-UNAM team 2010/2010/04/15

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Thursday 15th April 2010

Making of the petri boxes by melting the schott bottles in the microwave for about 7 minutes at 30% potency, then cool them a little into water until it’s bearable to feel the with the check, now add ampicillin (100mg/ml) per ml now getting a concentration of 100ug/ml in the medium, and for kanamycin (30mg/ml) the same and the final concentration is 30ug/ml.
I made a mistake duuhh… I labeled the boxes in the top instead of in the bottom.
Sterilize the boxes with fire and fill them, then close them with paraffin.

  • Extraction of the biopart:

From the 2009 Kit Plate 1, well 10H. I extracted the Firefly luciferase - luciferase from Photinus pyralis Ba_I712019, for this I added 15ul of water to the well and I extracted all and placed it in a tube labeled: 2009 Ba_I712019 H10 Firefly.
This biopart comes within the pSB1AK8 high copy number plasmid (100-300 copies per cell), and a length of 3426 bp, resistant to ampicillin and kanamycin.

PCR rtth (hot start):


  • 1º Reaction


................................1x....................4.5x
H2O.........................11.5ul..............51.7ul
Buffer 3.3x...............5ul...................27ul
Mg(Ac)2....................3ul..................13.5ul
dNTP’s......................2.5ul...............11.25ul
Oligo up-suffix...........2.5ul...............11.25ul
Oligo down-preffix.....2.5ul...............11.25ul
DNA..........................2ul...................---
Total.........................30ul................135ul


  • 2º Reaction


........................1x (ul) ........4x (ul)
H2O.................10.5...........47.25
Buffer 3.3x.......9................40.5
Rtth..................0.5.............---
Total................20..............87.75

I made in total 4 tubes for the first reaction, 2 for the PCR and one negative control with water instead of DNA and the positive one with a 2kb DNA of something else.
For the second reaction in one tube I made the mix but added the enzyme rttg at last.

  • For the whole hot start PCR there were 3 steps:

1) Place the tubes with rx 1 in the termocycler for 4:30 minutes at 95ºC for denaturalization.
1.5) Open the termocycler and add 20ul of the mix with the rtth enzyme to each tube.
2) 30 cycles:

  • 45 secs at 95º C for denaturalization
  • 45 secs at 60º for annealing
  • 1:40 minutes at 72º for elongation

3) 5 mins at 72º so whatever is going on finishes the hold at 4º.





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