IGEM:Paris Bettencourt 2012/Notebooks/Semantic group/day by day//2012/07/21: Difference between revisions

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(*) : calculated thanks to the following formula :
(*) : calculated thanks to the following formula :


<math>Weight_{insert} = ratio \times \frac{Size_{insert}}{Size_{vector}} \times Weight_{vector}</math>
<math>Mass_{insert} = ratio \times \frac{Size_{insert}}{Size_{vector}} \times Mass_{vector}</math>


ratio being 5 (5 times as many insert as vector).
ratio being 5 (5 times as many insert as vector).

Revision as of 08:20, 21 July 2012

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Making -20°C & -80°C stocks

  • I saved 750µL of O/N culture, in order to add 250µL of 60% glycerol sterile, in sterile tubes. I don't find them. I put the 750µL in normal eppendorf, at 4°C.

Miniprep of : pSC001a, pSC002a&b, pSC003a

  • I used 500µL of Lysis solution instead of 250µL
  • Recover plasmid in 70µL of water.
  • Nanodrop :
    • pSC001a : 186,4 ng/µL
    • pSC002a : 157,1 ng/µL
    • pSC002b : 144 ng/µL
    • pSC003a : 142,4 ng/µL

->the big amount of lysis solution did not perturb the miniprep.

Digestion of pSC001(a) & pSC002(a)

  • pSC001 : SpeI + PstI
    • DNA : 20 µL / 3
    • SpeI : 2 µL / 0
    • PstI : 2 µL / 0
    • buf. 10X : 4 µL / 2
    • water : 12 µL / 15
  • pSC002 : EcoRI + XbaI
    • DNA : 20 µL / 3
    • EcoRI : 2 µL / 0
    • XbaI : 2 µL / 0
    • buf. 10X : 4 µL / 2
    • water : 12 µL / 15

--> 30 min, 37°C water bath.

Migration on 1% agarose gel

Migration of 10µL of the digestions

Agarose gel of the previous digestion. E : EcoRI; P: PstI; X : XbaI; S : SpeI; Ø : no enzyme.

The bands are as expected, we can PCR purif the digestion (2 tubes of 30 µL)

PCR purif

The PCR purif is made with the promega kit, I recover dna in 30 µL of water.

  • pSC001 : 83 ng/µL
  • pSC002 : 76 ng/µL

-> good.

Ligation of pSC002 dig. + pSC003 dig.

  • vector (pSC002) : 1 µL (ie : 76ng)
  • insert (pSC003) : 37 µL (*)
  • buf. 10X : 1µL
  • T4 dna ligase : 1µL
  • water : 4 µL

(*) : calculated thanks to the following formula :

[math]\displaystyle{ Mass_{insert} = ratio \times \frac{Size_{insert}}{Size_{vector}} \times Mass_{vector} }[/math]

ratio being 5 (5 times as many insert as vector).

-> 22°C


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