IGEM:Paris Bettencourt 2012/Notebooks/Semantic group/day by day//2012/07/22: Difference between revisions

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===Result of the digestion===
===Result of the digestion===
I cut the bands out, in order to make a gel purification.
I cut the lower bands out, in order to make a gel purification.




Revision as of 14:13, 22 July 2012

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Result Ligation

Plate colonies?
Ligation pur +++
Ligation 1/10 ~280
positive transformation control (pSB1A2::R0011) +++
negative ligation control (pSC002 dig. ie. insert) 0
negative ligation control (pSC003 dig. ie. vector) ~240
negative transformation control (NEB only) 0

There is a background of self ligation, I expect that 1 over 10 bacteria have the wrong plasmid. So I make a PCR colony, and I make a culture of colonies I PCR.

  • Tubes L1->L12 are clones from ligation plates.
  • Tubes T-1->T-3 are clones from negative control plate (vector)

Here is the content of 1 tube of PCR :

  • VR (diluted 10 times from the stock tube) : 2,5µL
  • VF2 (diluted 10 times from the stock tube) : 2,5µL
  • PCR MM 2X : 25 µL
  • water : 20 µL

Here is the PCR pgm :

  • 95°C - 15' (to lyse cells too)
  • 95°C - 30"
  • 50°C - 30"
  • 72°C - 40"
  • 72°C - 10'
  • 4°C -[math]\displaystyle{ \infty }[/math]

-> 24 cycles

The culture tube are composed of 10mL of LB + 10µL Amp + 10µL Kan

Result of PCR

Agarose gel of the previous PCR. We can see that there are 5 over 10 samples that have not the good size. For further experiments, we take the samples L11 and L12 that seem ok

Miniprep L11 & L12

I minipreped these 2 samples, I recover in about 80µL of water (50µL and 30µL).

  • Nanodrop :
    • L11 : 114 ng/µL
    • L12 : 92 ng/µL

Digestion of L11 and L12

In order to obtain the final construction, I need to recover the insert from the construction I made, to insert it in the vector pSC001, already digested with PstI and SpeI.

  • L11 & L12 : XbaI + PstI
    • DNA : 20 µL / 3
    • SpeI : 2 µL / 0
    • PstI : 2 µL / 0
    • buf. 10X : 4 µL / 2
    • water : 12 µL / 15

--> 30 min, 37°C dry bath.


Result of the digestion

I cut the lower bands out, in order to make a gel purification.


Agarose gel of the previous digestion. P: PstI; X : XbaI.

Biobricks retrieval

Parts number Name Distribution/Plate/Well Backbone
BBa_I14034 P(Kat) 2012/2/13B pSB2K3
BBa_B0034 RBS 2012/1/2M pSB1A2
BBa_B0015 double terminator 2012/1/23L pSB1AK3

I'll transforme them later.

Transformation

What's in the tube DNA volume (µL) Cell volume (µL) Plates (LB + Amp)
positive transformation control (pSB1A2::R0011) 0,5 20 200µL
L11 1 20 190µL pur & 200µL 1/20
L12 1 20 190µL pur & 200µL 1/20
pSB2K3::I14034 0,5 20 190µL pur & 200µL 1/20
pSB1A2::B0034 0,5 20 190µL pur & 200µL 1/20
pSB1AK3::B0015 0,5 20 190µL pur & 200µL 1/20
negative transformation control (NEB only) 0 20 200µL on Amp, Kan(<<200µL), Amp+Kan

-> O/N 37°C

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